Molecular Scale Studies of Phosphorus Speciation and Transformation in Manure Amended and Microdose Fertilized Indigenous Vegetable Production Systems of Nigeria and Republic of Benin

Round 1

Reviewer 1 Report

This study combines synchrotron X-ray absorption spectrometry and chemical analysis to explore the changes of phosphorus forms in soil before and after planting Amaranthus cruentus and Solanum macrocarpon in Ina and Ilesha to determine the relationship between the availability and mobility of P and its form formation in soil. This research is important for both soil phosphorus conversion and Integrated Soil Fertility Management However, there are some problems, which may need revision before the paper could be considered for publication.

How are the sampling distributed, and how are the sampling distributed before planting and after final harvest? Why isn't it mixed into one sample after collecting multiple points in the same area? How to determine that the individual sampling is representative? In the materials and methods, the author described the detection in detail, however, the description is too tedious, and the length can be reduced appropriately. Line 57-58: What is AV？Is it short for Amaranthus cruentus? In lines 164-165 you mentioned that in SM, the pH drops significantly over time, except for T80. And eCEC increase in T0 and T40, the reduction in the T20 and T80. This is how to get change and the change in pH eCEC conclusion was positively associated with it? The author used P K-edge XANES spectroscopy to explore the forms of phosphorus in soil, including organic P, adsorbed P, and apatite P, but have never mentioned which LCF standard samples are, which is a very important point in XANES. In addition, XANES spectroscopy is different from chemical extraction methods. The standard sample determination is mostly an accurate substance rather than a certain species, so which substances did the author integrate as organic phosphorus or adsorbed phosphorus? If author refer to the results of other standards, how can the obvious errors brought by the instrument state prove that the small differences in lines 287-288 are accurate? Please clarify it. For X-ray Fluorescence, why did you choose RF_AT0Bp and RF_AT40Ah. Although both samples are RF_AT samples, but the urea content is different, why not choose RF_AT0Bp and RF_AT0Ah or RF_AT40Bp and RF_AT40Ah? Moreover, the paper writing needs substantially improvement. The whole manuscript needs to be checked on the grammar and logic thoroughly.

Author Response

Dear Reviewer,

COVER LETTER

Thank you for your comments, please find below, a point by point response to your comments.

Comment 1: How are the sampling distributed before planting and after final harvest?

Response: Each plot was replicated four times, and had area of 6m², 6m X 1m in Ina, Benin Republic, and 3m by 2m in Ilesha, Nigeria. The plots were designed to depict the common practice of growing the vegetables at both sites. Soil samples were randomly collected (as much as possible, we took the sample following a 'M' pattern) from 9 to 10 points on each plot before planting and after final harvest. This sampling procedure and distribution is now captured in lines 106 to 108 of the revised manuscript.

Comment 2: Why isn't it mixed into one sample after collecting multiple points in the same area?

Response: Soil samples from each plot was well mixed into a composite sample, and sub-sampled for analysis. This is stated in lines 108 to 109 of the manuscript.

Comment 3: How to determine that the individual sampling is representative?

Response: We believe that our sampling technique, as stated in the responses above shows that the individual samples are indeed representative enough. 

Comment 4: In the materials and methods, the author described the detection in detail, however, the description is too tedious, and the length can be reduced appropriately.

Response: This section is now revised to be more simple. Please see Section 2.1 (lines 126 to 160) of the manuscript.

Comment 5: What is AV? Is it short form for Amaranthus cruentus?

Response: Yes, it was. This has now been revised as AC throughout the manuscript, to stay consistent with the SM we used as an abbreviation for Solanum macrocarpon, the other vegetable.

Comment 6: In lines 164 to 165 (173-174??) you mentioned that in SM, the pH drops significantly over time, except for T80. And eCEC increase in T0 and T40, the reduction in the T20 and T80. This is how to get change and the change in pH eCEC conclusion was positively associated with it?

Response: We do not understand this question/statement. Can more clarity be provided? 

Comment 7: The author used P K-edge XANES spectroscopy to explore the forms of phosphorus in soil, including organic P, adsorbed P, and apatite P, but have never mentioned which LCF standard samples are, which is very important in XANES. In addition, XANES spectroscopy is different from chemical extraction methods. The standard sample is the mostly an accurate substance rather than a certain species, so which substances did the author integrate as organic phosphorus or adsorbed phosphorus?

If author refer to the results of other standards, how can the obvious errors brought by the instrument state prove that the small differences in lines 287-288 (297-298??) are accurate? Please clarify it.

Response: We used phytic acid as a representative compound for organic P phase, brushite, a poorly crystalline calcium phosphate as a reference for the adsorbed P phase, a highly crystalline calcium phosphate (apatite), and an Al and Fe phosphate mineral. All reference compounds used in this study has been used in other peer-reviewed articles from our laboratory. Necessary changes has been made in the manuscript to reflect the standard reference compounds we used. Please see lines 143 to 153.

With respect to the differences mentioned in lines 297-298, the Goodness of fit values (in Table 3) was approximately 0.002, suggesting a statistically significant fit. Also, we did refer to the shift in P species as marginal (see line297)

Comment 8: For X-ray Fluorescence, why did you choose RF_AT0Bp and RF_AT40Ah. Although both samples are RF_AT samples, but the urea content is different, why not choose RF_AT0Bp and RF_AT0Ah or RF_AT40Bp and RF_AT40Ah?

Response: We chose to compare those two samples due to the presence of apatite on the bulk XANES; our purpose was to probe for the Ca-P associations directly.

Comment 9: Moreover, the paper writing needs substantially improvement. The whole manuscript needs to be checked on the grammar and logic thoroughly.

Response: We have revised grammar, and also had two other native English speakers with published scientific articles help proofread the manuscript.

It is our hope that our responses have provided enough clarity and satisfies your concerns. Kindly let us know if you need any further information.

Best regards.

Reviewer 2 Report

The work by Kar et al. employs X-ray absorption spectroscopy to determine Phosphorus speciation in soils cultivated with different vegetables.
The paper is well written and shows a typical and interesting application of XANES spectroscopy to study mixtures of potentially unknown complexes.
I have a few minor comments and I think that the paper will be worth of publication provided that the results of the linear combination analysis will be presented with more quantitative details.

1- Why is "Amaranthus cruentus" abbreviated as (AV)?
2- Are low concentration of P associated with low fertility?
3- Line 55: remove the comma between "technique, to"
4- Line 127: 2 cm^2?
5- What is the energy resolution of the detector? Do the measurement include both K-alpha and K-beta lines?
6- Line 140: how many standards have been included? Which ones?
7- Line 167: significantly less --> significantly lower
8- Line 227: poutry --> poultry
9- It would be nice to see the experimental data shown in Fig. 6a and 7a compared among themselves and not only with the corresponding fits.
How much do they differ one from the other? I'd suggest adding a dedicated plot and calculating R-factors or similar among all the different couples of experimental spectra.
This point is in my opinion relevant, since it can provide information on the significance on the fit results.
Moreover, all the fits should be characterized by a goodness-of-fit index (chi squared, R-factor), Just as an example, spectra AT40BP and AT0AH look quite similar, but the percentage in the linear combinantion are quite similar and visually the latter looks slightly worse than the former.

Author Response

Dear Reviewer,

COVER LETTER

Thank you for your comments, please find below, a point by point response to your comments.

Comment 1: Why is "Amaranthus cruentus" abbreviated as (AV)?

Response: This has now been revised as AC, to stay consistent with the SM we used as the abbreviation for Solanum macrocarpon, the other vegetable.

Comment 2: Are low concentration of P associated with low fertility?

Response: Not when the soil is properly managed, however, this vegetable production practice is very intensive, and majority of producers in the region typically only add N fertilizer (as N source) and manure (as a P source), hence the risk of P mining. Soils of the SSA region are also typically formed from Kaolinite mineral and have a low inherent P fertility.

Comment 3: Line 55: remove the comma between "technique, to"

Response: Correction has been made in the revised manuscript.

Comment 4: Line 127: 2cm²?

Response: Correction has been made in the revised manuscript.

Comment 5: What is the energy resolution of the detector? Do the measurement include K-alpha and K-beta lines?

Response: The measurement included the P emission (both lines). The manuscript has been revised to reflect this. See lines 133-134.

Comment 6: How many standards have been included? Which ones?

Response: We used phytic acid as a representative compound for organic P phase, brushite, a poorly crystalline calcium phosphate as a reference for the adsorbed P phase, a highly crystalline calcium phosphate (apatite), and an Al and Fe phosphate mineral. All reference compounds used in this study has been used in other peer-reviewed articles from our laboratory. Necessary changes has been made in the manuscript to reflect the standard reference compounds we used. Please see lines 143 to 153.

Comment 7: significantly less --> significantly lower

Response: Correction has been made in the revised manuscript.

Comment 8: Line 227: poutry --> poultry.

Response: Correction has been made in the revised manuscript.

Comment 9: It would be nice to see the experimental daa shown in Fig 6a and 7a compared among themselves and not only with the corresponding fits. How much do they differ one from the other? I'd suggest adding a dedicated plot and calculating R-factors or similar among all the different couples of experimental spectra. This point is in my opinion relevant, since it can provide information on the significance on the fit results. Moreover, all the fits should be characterized by a goodness-of-fit index (chi-squared, R-factor), just as an example, spectra AT40BP and AT0AH look quite similar, but the percentage in the linear combination are quite similar and visually the latter looks slightly worse than the former.

Response: The objective of this study is to identify P species present in the soil before planting and after harvest, and to determine how individual treatments affects the availability and mobility of P in the soil. We figured that presenting our data as they are in figures 6a and 7a (comparing the data with the fits) serves that purpose well, and that comparing the experimental data among themselves would digress from that objective, and not necessarily improve the article. As the data is currently presented, we can infer how each vegetable and treatment influences soil P in each site and also predict its mobility.

With respect to the differences mentioned in the AT40BP and AT0AH spectra, we did characterize the fits by a goodness-of-fit index, the Goodness of fit values (in Table 3) typically ranged from 0.002 - 0.005, indicating a statistically significant fit.

It is our hope that our responses have provided enough clarity and satisfies your concerns. Kindly let us know if you need any further information.

Best regards.

Round 2

Reviewer 1 Report

Except for the following two comments, all replies are accepted.

Comment 6: In lines 187-188 of the revised manuscript, the author mentioned that the changes in eCEC positively correlated with the changes in pH in SM_RF, but comparing the changes in pH and eCEC in lines 171-174 and 186-187 are obviously not Positive correlation, please explain.

Comment 8: Now that all reference compounds have been used in other published articles, please cite that article to lines 144-146.

Author Response

Dear Reviewer,

Thank you for your comments, please find below, a point by point response to your comments.

Comment 6: In lines 187-188 of the revised manuscript, the author mentioned that the changes in eCEC positively correlated with the changes in pH in SM_RF, but comparing the changes in pH and eCEC in lines 171-174 and 186-187 are obviously not Positive correlation, please explain.

Response: As stated in lines 174-175 of the revised manuscript, we observed a trend where soils cultivated with Amaranthus cruentus (AC), generally had a pH reduction over time in both the Dry Savanna (DS) and Rainforest (RF) ecoregions (Tables 1 and 2). Also, from lines 188-190, we observed a near-similar trend where the eCEC reduced with time in all treatments of both the DS (except for T80) and RF (except for T0) ecoregions in the soils cultivated with AC. 

This observed trend is what lines 191 to 192 of the manuscript is referring to, and not necessarily a correlation. The sentence (in line 191 to 192) has now been rephrased to more appropriately refer to the observation as a trend, and not a correlation. Please see lines 191 to 194 of the revised manuscript.

Comment 8: Now that all reference compounds have been used in other published articles, please cite that article to lines 144-146.

Response: Necessary changes that cites the use of all reference compounds in other peer-reviewed articles have been made. Please see lines 145 to 146 of the revised manuscript.

We hope that our responses have provided enough clarity and satisfies your concerns. Kindly let us know if you need any further information.

Best regards