Addition of Phosphatases and Phytases to Mature Compost to Increase Available Phosphorus: A Short Study

Round 1

Reviewer 1 Report

The manuscript “Addition of Phosphatases and Phytases to Mature Compost to Increase Available Phosphorus” (agronomy-1488382) deals with the interesting topic of increasing the availability of phosphorous in compost by adding microorganisms and/or enzymes. The topic is of clear interest to the readers of Agronomy and the work seems to be innovative. Nevertheless, I have to report some major issue that must be addressed by authors before publication.

1) The manuscript require an extensive English editing. Sadly, it was really hard to understand the text sometimes.

2) The manuscript require an extensive editing of punctuation and orthography (i.e. there are lot of missing spaces, units errors in the text, specific names are sometimes in italic and sometimes not).

3) The manuscript require an extensive editing of references in the text (sometimes they are even reported as apex text) and in the list (please see journal instructions).

4) The manuscript require an extensive editing of figures and tables, with particular regard to captions.

5) Please explain how do you select the concentration used in the experiment to assess phosphate-solubilizing activity on compost. It is hard to understand why you used only one concentration to evaluate the different treatments and only then you used different concentrations with the “best” treatment. Please explain this in deep, referring also to literature, because this can be a bottleneck of your work hard.

6) I feel that some information about the compost used in the experiments should be added. I mean: origin, composition of the feedstock, chemical analysis. I guess that this is a first work, but I strongly recommend you to carry out also other trials on different compost since this is a really heterogeneous materials which characteristics depend on the feedstocks, process evolution, etc.

7) It is hard to me to understand the possible application of these treatments in real-scale composting. I feel that the paper is missing connection with real applications. Please, try to introduce this aspect in the introduction, discussion and conclusions.

Some minor comments:

• Keywords: delete the numbers
• L86-89: delete from introduction
• L174-177: please rephrase
• L189-191: please rephrase

In the light of these, I have to suggest the editor to push the authors solving all these major revisions before publication.

Author Response

Answers to Review 1

The manuscript “Addition of Phosphatases and Phytases to Mature Compost to Increase Available Phosphorus” (agronomy-1488382) deals with the interesting topic of increasing the availability of phosphorous in compost by adding microorganisms and/or enzymes. The topic is of clear interest to the readers of Agronomy and the work seems to be innovative. Nevertheless, I have to report some major issue that must be addressed by authors before publication.

The manuscript require an extensive English editing.

Answer:

This manuscript was edited in English professionally by the American Journal Experts, after this we modified some parts of the manuscript, so these parts were revised and the editing certificated is attached at the end of this file.

 

The manuscript require an extensive editing of punctuation and orthography (i.e. there are lot of missing spaces, units errors in the text, specific names are sometimes in italic and sometimes not).

Answer:

All changes were carried out as indicated along the manuscript.

 

The manuscript require an extensive editing of references in the text (sometimes they are even reported as apex text) and in the list (please see journal instructions).

Answer:

References were edited as indicated throughout the text.

The manuscript require an extensive editing of figures and tables, with particular regard to captions.

Answer.

All tables and figures were edited. A new table was added this table was numbered Table 1 so the rest of the tables were renumered.

Table 1. Physicochemical composition of mature cow manure compost.

 

Physicochemical composition	Values (%)
Total Nitrogen	1.955
Total Phosphorus	0.790
Carbon/Nitrogen Ratio	3.298
Organic Matter	11.158
Moisture Content	77.767
Ashes	11.165

 

 

 

 

Original Table 1 in Section 2.4, L127:

Table 1. Different treatments of P. aeruginosa ATC 15442 on mature compost

Treatment Code	Description-Composition
PhoEnz	Supernatant(crude enzymes) with phytases and alkaline, acid, and neutral phosphatases onPDB (phosphate-solubilizing enzymes(PhoEnz))
P. aeruginosa	P. aeruginosa ATC15442 as phosphate-solubilizing bacteria on PDB
Phy	Supernatant(crude enzymes) with phytases(Phy) on PHY
PhoEnz+ P. aeruginosa	Combination of PhoEnz (5% )and PSB (5%)
PhoEnz + P. aeruginosa+ Phy	Combination of PhoEnz (3.3%),PSB (3.3%) and Phy (3.3%)

 

Modified Table 1:

Table 2. Treatments of P. aeruginosa ATC 15442 on mature compost

Treatment Code	Description-Composition
PhoEnz	Enzymatic cocktail with phytases and alkaline, acid and neutral phosphatases on PDB
P. aeruginosa	P. aeruginosa ATC15442 as phosphate-solubilizing bacteria on PDB
Phy	Phytases on PHY
PhoEnz+ P. aeruginosa	Combination of 5% and 5%.
PhoEnz + P. aeruginosa+ Phy	Combination of 3.3%, 3.3% and 3.3%.
W	Deinoized water (control group).

 

Original Table 2 in Section 3.1; L 163;

Table 2. Specific activity of phosphate-solubilizing enzyme exudates for P. aeruginosa ATC15442 on PDB.

Enzyme type	Average Specific activity (U/mg protein)
Alkaline phosphatase	0.242 ±0.0086
Acid phosphatase	0.259 ±0.0098
Neutral phosphatase	0.161 ±0.0072
Phytase	0.005 ±0.0003

Modified Table 2:

Table 3. Enzymatic activity of P. aeruginosa ATC15442 on PDB.

Enzyme type	Average Enzymatic activity (U mg protein)
Alkaline phosphatase	0.242 ±0.0086
Acid phosphatase	0.259 ±0.0098
Neutral phosphatase	0.161 ±0.0072
Phytase	0.005 ±0.0003

 

Original Figure 1 in Section 3.2, L178;

 

Figure 1. Conversion of Po into Pi (Olsen P) on mature compost in a phosphate-solubilizing experiment, with deionized water as control(W), acid, alkaline, and neutral phosphatases and phytase on PDB (PhoEnz), inoculated P. aeruginosa (P. aeruginosa), and enzyme phytase(Phy). Different letters above the columns indicate significant differences between the means at a p<0.05.

 

Modified Figure 1:

Figure 1. Conversion of Po into Pi on mature compost in a phosphate-solubilizing experiment. Different letters in each column indicate significant differences according to Tukey´s test (p<0.05).

 

 

Original Figure 2 in section 3.3, L 206;

Figure 2. Pi (OlsenP) released from mature compost by enzymes, Control (W), enzymatic cocktail on PDB (PhoEnz), and inactive enzymatic cocktail on PDB (InacPho).

Modified Figure 2:

Figure 2. Pi released from mature compost by enzymatic treatments

 

Please explain how do you select the concentration used in the experiment to assess phosphate-solubilizing activity on compost. It is hard to understand why you used only one concentration to evaluate the different treatments and only then you used different concentrations with the “best” treatment. Please explain this in deep, referring also to literature, because this can be a bottleneck of your work hard.

Answer:

In section 2.4, L131; the following information was added:

" The treatments of phytase, phosphate-solubilizing enzyme, bacteria and their combinations were evaluated at 10% of phosphate-solubilizing activity according to the increment of the established percentages of PSB inoculation [9, 23]. On the other hand, this is one of the first evaluations to increase Pi in mature compost at 24-72 h ".

 

 

I feel that some information about the compost used in the experiments should be added. I mean: origin, composition of the feedstock, chemical analysis. I guess that this is a first work, but I strongly recommend you to carry out also other trials on different compost since this is a really heterogeneous materials which characteristics depend on the feedstocks, process evolution, etc.

Answer:

In section 2.3, L124; the following text was added:

"The physicochemical composition of the compost used is described in table 1"

so table 1 was added as described above.

It is hard to me to understand the possible application of these treatments in real-scale composting. I feel that the paper is missing connection with real applications. Please, try to introduce this aspect in the introduction, discussion and conclusions.

Answer:

In the Introduction Section L59, the following text was added:

"We used a PDB culture medium in the main experiments because their main components are easy available".

 

In the Introduction Section L83; the original text was changed

Original text:

"culture in potato dextrose broth (PDB) at the commercial scale";

was changed by the following text:

"culture in potato dextrose broth (PDB) was selected to allow further commercial scale".

In discussions section was added

"Enzyme cocktail treatment offers several advantages, mainly due to the easy reproduction of P. aeruginosa grown and low cost of the basic components of PDB".

In conclusions Section the following text was added:

"The use of PDB needs further investigations to confirm real scale production".

 

Some minor comments:

Keywords: delete the numbers

Answer:

All numbers were deleted.

 

L86-89: delete from introduction

Answer:

We made the changes.

 

L174-177: please rephrase

Answer:

The text was rephrase:

" PhoEnz 24 increase Pi compared with all the treatments and 20% compared to W.

PhoEnz+ P. aeruginosa and PhoEnz+ P. aeruginosa+ Phy showed no difference from W, which suggests interference caused by PSB that consumed the delivered P. This result is supported by Phy 72 and all PhoEnz (24, 48, and 72),which showed the highest percentages of Pi ( Figure 1)".

 

 

L189-191: please rephrase

Answer:

 The lines were removed because they did not refer to the results section and were placed on discussions section:

" The present work is one of the first to report the addition of PSB, phytases and phosphatases aiming the increase of Pi in mature cow manure compost

 

In the light of these, I have to suggest the editor to push the authors solving all these major revisions before publication.

 

Author Response File: Author Response.pdf

Reviewer 2 Report

Ortega-Torres et al., conducted a short study to evaluate the effects of phosphatases and phytases additions to compost to enhance plant-available phosphorus (Pi). The topic is of great interest, particularly while world phosphate rock reserves are limited and the agricultural community needs to find alternative strategies and ways to improve P use efficiency in different cropping systems. However, the current manuscript needs substantial work to make it worth publishing in Agronomy-MDPI. This reviewer does have a few general and specific comments for the authors regarding the current manuscript.

• The title does not reflect the study conducted such as no information is provided on microorganisms. Secondly, I recommend including “a short trial/study” in the title for more clarity.
• Abstract and graphical abstracts are very descriptive and do not convey the complete results. The authors should consider providing some correlations with total P, available P and organic P under different treatments in the abstract whereas should include some mechanism(s) in the graphical abstract to complete the story. The current graphical abstract is meaningless at least to this reviewer. The authors can benefit by including the % increase/decrease in Pi concentrations in compost due to the addition of enzymes along with actual Po values in compost. The same could be applied to yellow and brown circles to highlight changes.
• The authors use the term enzymatic cocktail at some points and not on others, therefore the authors need consistency for a better flow of ideas.
• The same comment for different abbreviations that are being cited in the manuscript. For instance, either available P or Pi, the same as for phosphorus or P?
• The authors included discussion in the results section and also have a separate discussion section. Therefore, authors should check which format should be followed as the current approach is a bit confusing.
• Include a working hypothesis at the end of the introduction section.
• The author might consider including a sketch of the current two experiments conducted to make it clear for the readers as the current text is not very clear at least to this reviewer.
• The authors should mention why the experiment was not repeated to validate the results and how one run could provide useful information especially when involving only 3 replications?
• The authors have measured total P and did not mention it in the results or discussion section? It would be great to include results to see what are the total P-reserves in compost and what portion is being made available by the different treatments? In fact, the authors should make a P budget about total P measured through acid digestion, Olsen P, and organic P among different treatments to see which treatment is more efficient. Reporting only Pi might not reflect the true picture due to different total P concentrations in the compost.
• Highlight what ±values mean in Table 2?
• L148: Olsen method for total P versus L135 for acid digestion?
• L166-L170: The authors need to keep discussion for discussion section or combine results and discussion section together for more clarity.
• L10: second most ?
• The sentence is incomplete to convey the message. Authors might want to say “……….. therefore the other countries largely depend on imports…..” or some text like this?
• L20: Include the implications of the result to complete the conclusion section. What are the perspectives of the results and this research?
• What do 1, 2, 3, 4 and 5 mean in keywords?
• L43: food chain2?
• L93: include the city and country name where the institute is located.
• L94: 24 hours to 24 h, apply the same comment to the rest of the manuscript wherever applicable.
• L105: 5 minutes to 5 min, apply the same comment to the rest of the manuscript wherever applicable.
• L113: Include the source of the spectrophotometer.
• L114: protein¹⁶?
• L123: Samples of 75-day mature………. the sentence is not clear.
• L124: were there rocks in the compost of stones?
• Mesh 0.29 m² m^-2?
• L174: Figure 1.?
• L174 – L177: Sentences are not clear to this reader. Rephrase the sentences for more clarity.
• L185: 20% increase seems misleading when compared to other treatments. For instance, the authors might check if W, P.ae, phytase or combination has the same 20% ratio compared to PhoEnz especially when authors state compared to all treatments.
• L194: The delivery of P ……………….. sentence is incomplete and needs authors’ attention.
• L196-L206: The authors want to include a discussion section in the results?
• There are several grammatical mistakes that need the authors' attention.

 

Author Response

Answers to Reviewer 2

 

The title does not reflect the study conducted such as no information is provided on microorganisms.

Answer:

In L51-59, microorganisms are described briefly, however the focus of the study was to use enzymes so that is why the word microorganisms is not included in the title.

Secondly, I recommend including “a short trial/study” in the title for more clarity.

Answer:

We added the word short study on the title.

 

Abstract and graphical abstracts are very descriptive and do not convey the complete results. The authors should consider providing some correlations with total P, available P and organic P under different treatments in the abstract whereas should include some mechanism(s) in the graphical abstract to complete the story. The current graphical abstract is meaningless at least to this reviewer. The authors can benefit by including the % increase/decrease in Pi concentrations in compost due to the addition of enzymes along with actual Po values in compost. The same could be applied to yellow and brown circles to highlight changes

Answer:

Original graphical abstract:

Modified graphical abstract:

 

The authors use the term enzymatic cocktail at some points and not on others, therefore the authors need consistency for a better flow of ideas.

Answer.

 All changes have been made as indicated throughout the manuscript.

 

The same comment for different abbreviations that are being cited in the manuscript. For instance, either available P or Pi, the same as for phosphorus or P?

Answer.

All changes have been made as indicated throughout the manuscript

 

The authors included discussion in the results section and also have a separate discussion section. Therefore, authors should check which format should be followed as the current approach is a bit confusing.

Answer:

All the changes were carried out as indicated in manuscript

 

Include a working hypothesis at the end of the introduction section.

Answer

 At the end of introduction we eliminated

"In this study, the addition of phosphorus-solubilizing enzymes isolated from Pseudomonas aeruginosa PSB to mature compost for increasing the concentration of Pi was evaluated".

and added the following sentence for hypothesis:

"According to this the following hypothesis is stated. The addition of phosphate-solubilizing enzymes on mature compost will increase Pi compared to conventional composting".

 

The author might consider including a sketch of the current two experiments conducted to make it clear for the readers as the current text is not very clear at least to this reviewer.

Answer:

 We did not make a sketch however we change the titles of the section 2.3 and 2.4

Original

2.3 Experiment to assess phosphate-solubilizing activity on compost.

 Was change by

2.3 Assesment of phosphate solubilizing activity of enzymes, bacteria and combination to increase Pi on mature compost.

Original

2.4 Experiment to increase the phosphate-solubilizing activity on compost.

Was change by:

2.4 Assesment of PhoEnz in higher percentages to increase Pi on mature compost.

 

The authors should mention why the experiment was not repeated to validate the results and how one run could provide useful information especially when involving only 3 replications?

Answer:

Three replications are statistically significant, and we presented articles with same replications: Feng et al., [9] or Parastesh et al., [23].

 

The authors have measured total P and did not mention it in the results or discussion section? It would be great to include results to see what are the total P-reserves in compost and what portion is being made available by the different treatments? In fact, the authors should make a P budget about total P measured through acid digestion, Olsen P, and organic P among different treatments to see which treatment is more efficient. Reporting only Pi might not reflect the true picture due to different total P concentrations in the compost.

Answer:

In section 2.3, the following text was added:

"Pi percentages were calculated on reference to Total P".

 

 

Highlight what ±values mean in Table 2?

Answer:

We eliminated the values that correspond to standar deviation.

 

L148: Olsen method for total P versus L135 for acid digestion?

Answer:

The Olsen method was used for Pi, while the digestion in nitric acid was used for Total P;

We rephrased:

L148 " Compost triplicate samples were quantified for Pi by the Olsen method and for total P by digestion in nitric acid".

 

L166-L170: The authors need to keep discussion for discussion section or combine results and discussion section together for more clarity.

Answer.

The changes were carried out as indicate in the manuscript.

 

L10: second most ?

Answer:

We eliminated the word "most".

 

 The sentence is incomplete to convey the message. Authors might want to say “……….. therefore the other countries largely depend on imports…..” or some text like this?

Answer:  

Original L41-42

"For food safety, this outlook is not hopeful, since 97% of countries depend on imported phosphate fertilizers"

was changed by:

"For food safety, this outlook is not hopeful, as this may compromise agriculture in the near future"

 

L20: Include the implications of the result to complete the conclusion section. What are the perspectives of the results and this research?

Answer:

In conclusions section, the following text was added:

"The use of PDB needs further investigations to confirm real scale productionand the final product needs to be evaluated on agriculture".

 

What do 1, 2, 3, 4 and 5 mean in keywords?

Answer.

We eliminated the numbers in keywords.

 

L43: food chain2?

Answer.

We wrote the number in the correct format.

 

L93: include the city and country name where the institute is located.

Answer:

We added the city and country to the institute

 

L94: 24 hours to 24 h, apply the same comment to the rest of the manuscript wherever applicable.

Answer:

The changes were carried out as indicate in the manuscript

 

L105: 5 minutes to 5 min, apply the same comment to the rest of the manuscript wherever applicable.

Answer:

The changes were carried out as indicate in the manuscript

 

L113: Include the source of the spectrophotometer.

Answer:

We added the source in the following text:

"using a UV-Vis spectrophotometer (SP-UV 1100, DLAB)"

 

L114: protein¹⁶?

Answer.

We wrote the number in the correct format.

 

L123: Samples of 75-day mature………. the sentence is not clear.

Answer:

We eliminated "75 day".

 

L124: were there rocks in the compost of stones?

Asnswer:

We changed "rocks " by "stones".

 

Mesh 0.29 m² m^-2?

Answer:

We changed the units to "0.29 mm".

 

L174: Figure 1.?

Answer:

We changed the position of the "Figure 1" and the end of the paragraph.

 

L174 – L177: Sentences are not clear to this reader. Rephrase the sentences for more clarity.

Answer:

The text was rephrase:

"PhoEnz 24 increased the Pi by 20% in compost compared to W.

PhoEnz+ P. aeruginosa and PhoEnz+ P. aeruginosa+ Phy showed no difference from W, which suggests interference caused by PSB that consumed the delivered P. This result is supported by Phy 72 and all PhoEnz (24, 48, and 72),which showed the highest percentages of Pi ( Figure 1)".

 

L185: 20% increase seems misleading when compared to other treatments. For instance, the authors might check if W, P.ae, phytase or combination has the same 20% ratio compared to PhoEnz especially when authors state compared to all treatments.

Answer:

Original:

"PhoEnz 24 increased the Pi by 20% in compost compared to all treatments"

L185 was change by:

"PhoEnz 24 increase Pi compared with all the treatments and 20% compared to W".

 

L194: The delivery of P ……………….. sentence is incomplete and needs authors’ attention.

Answer:

The change was made to L194

"Pi delivery was not proportional between PhoEnz treatments".

 

L196-L206: The authors want to include a discussion section in the results?

Answer:

We changed t text to discussions section.

 

There are several grammatical mistakes that need the authors' attention.

Answer:

All the changes were carried out as indicate in the manuscript.

Author Response File: Author Response.pdf

Round 2

Reviewer 1 Report

Dear authors, 

Thanks for having addressed all the comments. I feel that the paper now is overall worthing publication.

Best regards

Author Response

No changes were done as no observations were made.

Reviewer 2 Report

I would like to thank the authors for revising the article; however, there are still some minor corrections required. For instance, "available P or P availability" instead of P available" in L14. L20 is merely a result and not the conclusion. The authors should include a statement that the current study demonstrated the importance of adding .................. to enhance P availability in mature compost; however, further studies are required to confirm the results and practical applications...................... Sentences in L18-19 need better structure. The same comment of L52-56. As indicated earlier, the authors need consistency for Pi versus P, P versus Po, check the abbreviations form abstract till conclusion section. See L105 for PHY ()? It was suggested to explain what ±values mean in the table. Do not delete the values as those show the variability and just explain these in the Table footnote or title. The title of Table 2 is missing? h not hrs. L105-151: Pi percentages were calculated in reference to Total P Table 2. Treatments of P. aeruginosa ATC 15442 on mature compost? I think Table 2 explains the treatments? Include what error bars mean in all figures. Include the number of reps for clarity for each mean. Figure 1 title is a bit misleading as it is not the conversion of Po into Pi as there may be other factors. Better to write observed Po and Pi in ....................... The same comment for other figures. What does * mean in L235? Check L217-L218 and L257-L260, avoid redundancy in the text. The authors should include what are the several advantages offered by enzyme cocktail addition to mature compost. L576-L280 should be in the conclusion section and authors should also include that "this was a pilot short study and further research is required to validate the study output and..............". Overall, the authors need to strengthen the discussion section as there is no information how the treatments actually enhanced Pi in mature compost through which mechanism? Reporting only previous studies is not enough and the authors can include what would be the mechanism behind such changes and how that could be affected by different sources of manure or compost? 

Author Response

Comments for reviewer 2

In the grade of English, the reviewer says that an extensive English revision is needed.

The paper was edited by the American Journal Experts in a first version, but many changes have been done to the MS. The editing Certificate is attached.

 

I would like to thank the authors for revising the article; however, there are still some minor corrections required.

For instance, "available P or P availability" instead of P available" in L14.

Answer: The term was changed to:

"we evaluated the mineralization of P in mature compost",

 

 L20 is merely a result and not the conclusion.

The authors should include a statement that the current study demonstrated the importance of adding .................. to enhance P availability in mature compost; however, further studies are required to confirm the results and practical applications......................

Answer: We eliminated the L20;

and we added the following text:

"The current study demonstrated the importance of adding the cocktail to enhance Pi in mature compost; however, further studies are required to confirm the results and practical applications".

 

Sentences in L18-19 need better structure.

Answer:

L18-19 We changed the structure to:

"Second, we evaluated the mineralization of P in mature compost when inoculated with P. aeruginosa ATC 15442, phytases, a cocktail of phosphate-solubilizing enzymes and their  combinations . Finally, we evaluated different concentrations of the cocktail trying to release more P in the compost"

 

The same comment of L52-56.

Answer:

We changed the following text in L52-56 to:

"Currently, it has been estimated that 40% of P absorbed by plants worldwide comes from organic waste decomposition in soil [7,8].

An ecological, economical and practical way to recycle organic P (Po) in wastes is through composting; however, composting generates low  conversion rates of Po into inorganic P (Pi). One way to increase Pi in compost is to add phosphate-solubilizing microorganisms (PSM) [7]".

 

 As indicated earlier, the authors need consistency for Pi versus P, P versus Po, check the abbreviations form abstract till conclusion section.

Answer:

We revised as indicated, and also added in discussion section, the following text:

"In soil, P is present in different chemical forms such as Pi available for plants or microorganisms, the immobilized P is solubilized through organic acids exudated by PSM; and the Po is mineralized through enzymes exudated by PSM.  Once the immobilized P and Po have been solubilized, P becomes Pi."

 

 See L105 for PHY ()?

Answer:

We did not make any change as before in the same Line it is explained what PHY means:

 " phytase production medium (PHY) containing sodium phytate"

only was added the reference "[15]".

 

It was suggested to explain what ±values mean in the table. Do not delete the values as those show the variability and just explain these in the Table footnote or title.

Answer:

We corrected the delete of the values and  added the next explain:

"Values (±) represent the standard deviation".

 

The title of Table 2 is missing?

Answer:

The title is in L-206

 

h not hrs.

Answer:

The correction was made as indicated.

 

L105-151: Pi percentages were calculated in reference to Total P

Answer:

Original L105:

"Pi percentages were calculated in reference to Total P"

Modified to:

"Pi values were reported as percentages of Total P".

 

 Table 2. Treatments of P. aeruginosa ATC 15442 on mature compost? I think Table 2 explains the treatments?

Answer:

Original text:

"Table 2. Treatments of P. aeruginosa ATC 15442 on mature compost"

Modified text:

"Table 2.Phosphate-solubilizing combination of  P. aeruginosa ATC 15442 applied on mature compost"

 

Include what error bars mean in all figures.

Answer:

In each figure we added the following text:

"Error bars are the standard deviation

 

Include the number of reps for clarity for each mean.

Answer:

This is explain on the Materials and Methods Section; L136, L169 and  L187

so no change was made.

 

 Figure 1 title is a bit misleading as it is not the conversion of Po into Pi as there may be other factors. Better to write observed Po and Pi in ....................... The same comment for other figures.

Answer:

We changed the titles of the figures to:

"Figure 1. Pi obtained by phosphate-solubilizing combination of  P. aeruginosa ATC 15442  applied on mature compost"

"Figure 2. Pi obtained by phosphate-solubilizing enzymatic cocktail and inactivated cocktail applied on mature compost"

 

What does * mean in L235?

Answer:

We erased the symbol "*".

 

Check L217-L218 and L257-L260, avoid redundancy in the text.

Answer:

we changed L-217-218, to the following text:

"The phosphate-solubilizing activity of different treatments was evaluated by measuring Pi in mature compost. PhoEnz treatments showed an increment in Pi presence compared with all the treatments and 20% compared to W. Interestingly, treatments PhoEnz+ P. aeruginosa and PhoEnz+ P. aeruginosa+ Phy showed no difference from W which suggests interference between enzymes and the bacteria, on the other hand, treatment Phy 72 showed a Pi increment, which is only enzymes. "

 

We changed L257-L260, to the following text:

"The phosphate-solubilizing activity of different treatments was evaluated by measuring Pi in mature compost. PhoEnz treatments showed an increment in Pi presence compared with all the treatments and 20% compared to W. Interestingly, treatments PhoEnz+ P. aeruginosa and PhoEnz+ P. aeruginosa+ Phy showed no difference from W, on the other hand, treatment Phy 72, showed a Pi increment, which suggests interference between enzymes and the bacteria".

 

 The authors should include what are the several advantages offered by enzyme cocktail addition to mature compost.

Answer:

In L56-60;

We reference the advantages of the enzyme cocktail,

and we added in Conclusion Section, the following text:

"The phosphate-solubilizing enzymatic cocktail application has the advantage of increasing Pi on mature compost acting directly over Po substrate avoiding the competence between native and exogenous microorganisms on the compost. We think that the enzymatic cocktail methodology could be used successfully in the same manner in other sources of compost".

 

 L576-L280 should be in the conclusion section and authors should also include that "this was a pilot short study and further research is required to validate the study output and..............".

Answer:

We added in Conclusion Section, the next text:

"This was a pilot short study which represents an alternative to P transformation during composting, however we only released 12.7% of Total P hence further research is required to validate the study".

 

Overall, the authors need to strengthen the discussion section as there is no information how the treatments actually enhanced Pi in mature compost through which mechanism?

Reporting only previous studies is not enough and the authors can include what would be the mechanism behind such changes and how that could be affected by different sources of manure or compost? 

Answer:

We added in Discussion Section the next paragraph:

"In soil, P is present in different chemical forms such as Pi available for plants or microorganisms, the immobilized P  is solubilized through organic acids exudated by PSM; and the Po is mineralized through enzymes exudated by PSM.  Once the immobilized P and Po have been solubilized, P becomes Pi. Po has been reported to be present in several organic forms in manure compost such as phytic acid, phospholipidsa and nucleic acids. Po has been reported to be present in several organic forms in manure compost such as phytic acid, phospholipidsa and nucleic acids [24-26]. These molecules are substrates for enzymatic activity specially phytic acids represent about the 80% of organic waste, however, their links are very difficult to break. Phytases are the only enzymes that convert Po present in phytic acid into Pi. On the other hand, phosphatases manage the convertion of the other biological forms of Po [27].

The mechanism by which phytases and phosphatases mineralize Po depend mainly on the presence of the organic forms, working as enzymatic substrates. As we know, most of the organic wastes and therefore, compost processes contain phytic acid, phospholipids and nucleic acids, suggesting that this Pi release mechanism is suitable through compost processes with different organic matter sources".

 

Author Response File: Author Response.pdf