Biochemical and Microbiological Soil Effects of a Biostimulant Based on Bacillus licheniformis-Fermented Sludge

Round 1

Reviewer 1 Report

The manuscript presents interesting data. However, the writing is weak. I have annotated and attached the PDF. The abstract must be rewritten. MM section deserves more attention as most of the analysis has been referred to earlier studies. Results section has irrelevant methods details which must be shifted to relevant sections.

Comments for author File: Comments.pdf

Author Response

2.6. Statistical analysis

Did data follow the normality assumption?

Answer:  yes

Changes in Results

3.1.  Characterization of biostimulant products

              - description of FS, IFS and SFS goes to section 2.1

               - Proteomic charaterization: the initial lines (Given that the microbial community that conforms sludge includes the genus Bacillus,  the basal expression of Bacillus´proteins in sludge was compared  by mass spectrometry with that after fermentation) go to section 2.3.2.

3.2.1 . Initial paragraph in this section goes to 2.5.1

3.2.4. Effects on soil bacterial community diversity

 Te sentence:  Changes produced in soil bacterial biodiversity were studied through a metabarcoding analysis performed using the 16S rRNA marker, goes to section 2.5.2

Changes in figures and tables

- different figures corresponding to each enzimatic activities has been mentioned as A y B

-  legends: commas have been changed to points in doses and  p/p has been changed to w/w 

-  table 3: Decimals have been unified and commas have been changed to points

Changes in Discussion

4.1. Referee suggest that the following  paragraph is a repetition of results : as repetition It can be noted that the fermented sludge has an average organic matter content of 71.26 ± 0.31 w/w (table 1). Regarding its chemical composition, its content in N (5.63 ± 0.01 w/w, table 1) and P (17,663.19 ± 0.51 mg Kg-1, table 1) is remarkable, as well as its composition in heavy metals is below the limit values established by Spanish legislation for the use of this product in the agricultural sector (RD 1310/1990, of October 29) (table 1).

However we consider that it is relevant to highlight that the product composition is according to Spanish legislation for the use of this product in the agricultural. Anyway, we have deleted numbers.

4.2.2 Changes in soil at microbiological level

We do not understand why genus (Bacillus genus) is highlighted in yellow

 

Reviewer 2 Report

Dear Authors

 

your manuscript need signficant improvement in the language and fluency of the text. The text is too much descriptive so making is very long and boring. try to make it compact and more attractive. Reduce the length to half. 

Comments for author File: Comments.pdf

Author Response

Title: has changed to

A biostimulant based on Bacillus licheniformis-fermented sludge: biochemical and microbiological effects on soil

Abstract:

The abstract has been changed according to the referee's suggestion, it has been made more compact.

      Question 1. Efficiency goes to nutrients or to crop?

Answer: efficiency is related to absortion of nutrients. To avoid misunderstanding efficiency has been removed.  

       Question 2. Don’t describe them:

Answer: the description is related to the three products obtained in the fermentation process, that are the core of the study in present work. Thus we consider that their description in the abstract must be included to give clear information about the work.

Introduction:

       Question 1. The introduction has been shortened according to referee´s suggestion.

        Question 2. we do not agree that this text is not important; it gives specific information on the way to obtain new biostimulants, mainly from the point of view of their economic viability. Such as type of organic source, absence of toxics, local and focused production, not dispersed, etc.

However, we have rewritten the text following the referee´s suggestion.  Following the referee suggestion, the reference Aghaye Noroozlo et al., 2019; Souri and Bakhtiarizade, 2019  have been included.

        Question 3. we agree with the referee, the paragraph has been removed

         Question 4. we agree with the referee, the paragraph has been compacted in half.

Materials and methods:

Question 1. section 2.1. Obtaining the biostimulant

The sentence “the humidity of every resulted products and of raw sludge as control product were matched at 70 g L-“

was changed to “the humidity of the three biostimulants were matched at 70 g L-“

Humidity has been equalized in the different biostimulants through water evaporation for two reasons.

1. To have stable products, avoid microbiological growth
2. To have uniformity in matter content.

Question 2. 

section 2.3. Biological characterization of fermented products

The subtitle has been changed to: 2.3. Microbial and protein secreted characterization of fermented product

They have been briefly evaluated microbiological parameters (Bacillus concentration) and sequencing and analysis of proteins excreted by Bacillus.

Results.

    Question 1.

As suggested by the referee the subtitle of section 3.2.1. Changes in soil at biochemical level has been changed to 3.2.1. Soil biochemistry properties

- the paragraph (Soil enzymes were monitored during 28 days after aplication of the products in or-der to obtain a global vision of how the sludge-based biostimulants was acting in soil at biochemical level……) has been moved to MM. as also suggested by referee 1

      Question 2

The subtitle of section 3.2.2 Changes in soil at microbiological level, has been changed to  3.2.2 Soil microbiological characterization

    Question 3. The following paragraph has been deleted:

We have previously described that applying subtilisin, an endoprotease from Bacil-lus sp, induced interesting results on soil microbial biodiversity stimulating specific PGPR [19]. Therefore, it is to be expected that the application of a fermentation broth that contains not only the mentioned subtilisin but also a broad spectrum of enzymes, would lead to interesting changes in soil microbial biodiversity. In order to confirm our hypthesis bacterial biodiversity was analyzed in soil samples treated with the sludge-derived products at 0.5% w/w which was the dose of biostimulants that produced the highest stimulation in soil.

          Question 4. referee comment This is not results:

answer: the results of point 3.2.2. are shown in the following subtitles. There was a numbering error and 3.2.4 and 3.2.5 have been modified; Now these are 3.2.2.1 and 3.2.2.2 respectively.   These two sections are the subtitles of 3.2.2.

Discussion

Question 1

In section 4.1 . Characterization of biostimulant products

Changes have been made according to referee suggestion.

      -Subtitle in section 4.2 has been changed to 4.2. Soil biostimulant capacity of Biostimulants

    - Subtitle in section 4.2.1 has been changed according to referee’s suggestion to 4.2.1.Soil enzymatic activities.

   - ‘to a greater or lesser extent’ was deleted

All other minor points have been corrected according to referee´s suggestion.

Reviewer 3 Report

In this study, slaughterhouse wastewater sludge was used for fermentation，a new biostimulant has been devel-oped, through its fermentation with Bacillus licheniformis a plant growth-promoting bacteria (PGPB). These results show the fermentation process with B. li -cheniformis as an interesting option for the total valorization of activated sewage sludge. This is a very interesting study. However, heavy metals and antibiotics in a new biostimulant are unavoidable environmental risk factors for slaughterhouse wastewater sludge. This study only measured the content of heavy metals, and there was no research on antibiotics, and antibiotics often play an important role in the fermentation process, which will affect the activity of microorganisms, but there is no relevant measurement results and discussions in the article.

Author Response

We agree that it is very important to know the content of emerging contaminants, such as antibiotics, of a product that is going to be applied to the soil.

Although not included in the article, content of relevant antibiotics used for animal has been analyzed in the final fermented sludge and their concentration was not quantifiable.

The sludge comes from slaughterhouse wastewater that has been biologically purified in stirred bach reactor (SBR) through microbial growth processes, resulting in sludge, which is formed mainly by this microbial consortium. Microbial growth reveals that antibiotic concentrations, if any, are minimal. The sludge is then used as a fermentation source for Bacillus, which again shows optimal growth.

In any case, we are proposing a recovery process that is applicable to any sludge, and that could solve the environmental problem that they generate. However, in order to use a certain sludge as a raw material for this process, it is necessary to submit it to a regulatory control adapted to the regulations of the country where it is going to be applied.

Round 2

Reviewer 1 Report

The authors have addressed my comments. Therefore, I recommend accepting the manuscript in its current form.

Author Response

I appreciate your review

Reviewer 2 Report

Dear Authors,

It has been well revised and no further revision is needed

Author Response

I appreciate your review