Effect of Inoculum Concentration on the Degradation of Diesel 2 by a Microbial Consortium

Round 1

Reviewer 1 Report

General Comments:

In this experiment, the effect of inoculation concentration on the degradation of diesel 2 and the growth of bacteria in systems were studied in an aerated and stirred tank bioreactors.But the purpose of this study has not been clearly explained. Research about the effect of microbial concentration on degradation have been commonly explored.The novelty of this experiment may be the role of functional microflora BIOT PD001 in the degradation process, but the description of it in this paper is quite rare. Only three parameters(BOD₅, the number of bacteria and the concentration of total fats) were measured. The data seemed to be insufficient to support the conclusion.In results,about the conclusion that there is a linear relationship between inoculum concentration and removal efficiency as well as BOD₅, need to be verified.

Specific Comments:Including but not limited to following

Abstract:

line 104 :Information about BIOT.PD001 appears in the Materials section seems more reasonable

Line116:Whether ‘Rhodococcus is capable of reducing more than half the sulfur content in Diesel ’ is related to this experiment ?Ensure content relevance of references

Introduction：

line148:Lack of description of the reactor

Line150: ‘Minimum Davies Medium’ Specific description or annotation references

Line 159 Biomass Or microbial populations？

Results:

Line 194  Repeated ‘Inter-groups’

Line 205 Where is a?

Line 213 Non-English content appears in the table, and Figures 1-4 have the same problem

Line 234 contour map does not seem to be correct

Author Response

Thank you very much dear colleague, for the comments provided. I send the answers to each comment.
best regards

_________________________________________________________________________________

in this experiment, the effect of inoculation concentration on the degradation of diesel 2 and the growth of bacteria in systems were studied in an aerated and stirred tank bioreactors.But the purpose of this study has not been clearly explained.

Ans. Justification explanation improved

Research about the effect of microbial concentration on degradation have been commonly explored.

Ans. The novelty of this experiment may be the role of the functional microflora BIOT PD001 in the degradation process, but the description of it in this paper is quite rare.

The novelty of this experiment may be the role of functional microflora BIOT PD001 in the degradation process, but the description of it in this paper is quite rare. Only three parameters(BOD₅, the number of bacteria and the concentration of total fats) were measured. The data seemed to be insufficient to support the conclusion.In results,about the conclusion that there is a linear relationship between inoculum concentration and removal efficiency as well as BOD₅, need to be verified.

Specific Comments:Including but not limited to following

Abstract:

line 104 :Information about BIOT.PD001 appears in the Materials section seems more reasonable

Line116:Whether ‘Rhodococcus is capable of reducing more than half the sulfur content in Diesel ’ is related to this experiment ?Ensure content relevance of references

Ans. A more recent reference was placed in which Rodococcus acts in consortium with other bacteria and is equally efficient.

Introduction：

line148:Lack of description of the reactor

Ans.Bioreactor description expanded

Line150: ‘Minimum Davies Medium’ Specific description or annotation references

Ans. MMD components were placed

Line 159 Biomass Or microbial populations？

Ans.Biomass production is mentioned there as a synonym for microbial growth, the term was changed to microbial growth.

Results:

Ans. Tables and figures were improved

Line 194  Repeated ‘Inter-groups’

Ans.corrected

Line 205 Where is a?

Ans.corrected

Line 213 Non-English content appears in the table, and Figures 1-4 have the same problem

Ans.The translation of the words in Spanish was made

Line 234 contour map does not seem to be correct

Ans.Removed contour map and included multiple regression plot to better explain effect of inoculum concentration

Reviewer 2 Report

The submitted manuscript submitted by Otiniano et al. describes the influence of different inoculum concentration of diesel on the growth rate of a selected bioconsortium obtained from activated sludge. The author found that with increasing diesel concentration the specific growth rate of the biomass increases except for the highest inoculum concentrtuon. Overall, the authors were not able to state why this investigation was required? The finding the inoculum concentration in a batch process is influencing the biomass growth is trivial. So why must that be investigated and published? The manuscript has several weaknesses in style, layout and fundamental mistakes in the set-up. Therefore, I recommend to reject the manuscript. 

Some stylistic and layout pitfalls: 

1.     Misleading connection between first author and corresponding author

2.     Several times using “on the other hand” (line 81, 100, 116, 124 and so on)

3.     Several times unclear or doubling use of abbreviation (line 99 & 129; line 145 unclear abbreviation; line 304

4.     Language mixture in Tables and Figures!

5.     Before presenting Tables and Figures please explain them briefly in a text. Very often the text is below Tables and Figures and must be moved above.

6.     Bacterial species must be italic written

7.     The reactor set-up and condition should be present as text and more detailed description is required.

8.     Excel graphs should be polished and minimised to the main content allowing to extract only the results. 

Selected fundament mistakes:

1.     In the manuscript the diesel concentration is only provided in percentage. Thereby the reader has no information on the carbon concentration of the samples. This information is very important and could be provided in terms of COD and/or TOC. Since the authors discuss later growth rates at least the TOC concentration is required to allow any conclusion about bacterial growth. 

2.     From the presented results is it obvious that only two parameters were measured namely CFU (coliform forming unit) and the BOD₅. Again, more detailed investigation is necessary to allow any conclusion on the growth of the biomass in the reactors. Bacterial growth needs at least organic  carbon, a nitrogen source, phosphorus and some important trace metals. No further information is provided in the material and method section about the N and P source. Therefore, it remains unclear whether the growth rate might be limited with 10 % v/v diesel inoculum. However, it might also be that with higher concentration toxic effect of diesel or microbial competition for carbon decrease the growth rate. The authors are requested to clearly state the composition of the influence medium and loading rates. 

3.     The removal of diesel was only measured at the end of the batch tests. Therefore, it remains in doubts how to interpret data obtained from BOD₅ measurements only. The limited BOD₅ for 2% v/v diesel inoculum can be either a result of carbon limitation or due to other not investigated reasons. However, carbon limitation might a very expectable reason.

4.     The determination of the growth rates should be presented at least in a supplement file. The authors stated that they have performed all experiments in triplicate. Thus, the growth rate can be also reported with a standard deviation (SD). This is also true for the removal efficiency. 

5.     I did not understand the requirement to perform statistical analyses such as ANOVA and Duncan’s test. It only demonstrates that the removal efficiencies are true. In fact, a similar outcome would be simply obtained using SD.

6.     Is there any reason to provide four digits (66.6667)? What is the meaning? Does it make your determined results more precise? 

7.     Result interpretation in the discussion occurs very theoretical due to missing measurements.

Author Response

Thank you very much dear colleague, for the comments provided. I send the answers to each comment.
best regards

_______________________________________________________________

The submitted manuscript submitted by Otiniano et al. describes the influence of different inoculum concentration of diesel on the growth rate of a selected bioconsortium obtained from activated sludge. The author found that with increasing diesel concentration the specific growth rate of the biomass increases except for the highest inoculum concentrtuon. Overall, the authors were not able to state why this investigation was required?

Ans.  The reason for the investigation is better explained in the justification

The finding the inoculum concentration in a batch process is influencing the biomass growth is trivial. So why must that be investigated and published? The manuscript has several weaknesses in style, layout and fundamental mistakes in the set-up. Therefore, I recommend to reject the manuscript. 

Some stylistic and layout pitfalls: 

1. Misleading connection between first author and corresponding author

Ans. The main author of the manuscript is Dr. Otiniano.

2. Several times using “on the other hand” (line 81, 100, 116, 124 and so on)

Ans. was corrected

3. Several times unclear or doubling use of abbreviation (line 99 & 129; line 145 unclear abbreviation; line 304

Ans. Repeated terms were corrected, the Biochemical Oxygen Demand abbreviation is retained, since it is an abbreviation that is mentioned as such in texts and articles and is accepted in the field of science.

4. Language mixture in Tables and Figures!

Ans. was corrected

5. Before presenting Tables and Figures please explain them briefly in a text. Very often the text is below Tables and Figures and must be moved above.

Ans. The description of results was placed before the tables and figures.

6. Bacterial species must be italic written

Ans. All scientific names are italicized.

7. The reactor set-up and condition should be present as text and more detailed description is required.

Ans. A more detailed description of the bioreactor was made

8. Excel graphs should be polished and minimised to the main content allowing to extract only the results. 

Ans. graphics have been improved

Selected fundament mistakes:

1. In the manuscript the diesel concentration is only provided in percentage. Thereby the reader has no information on the carbon concentration of the samples. This information is very important and could be provided in terms of COD and/or TOC. Since the authors discuss later growth rates at least the TOC concentration is required to allow any conclusion about bacterial growth. 

Ans. In this case, diesel 2 is used as the only source of carbon, whose use or degradation is measured indirectly by measuring bacterial growth and by reducing the biochemical oxygen demand. COD and TOC are considered as limitations due to lack of resources.

2. From the presented results is it obvious that only two parameters were measured namely CFU (coliform forming unit) and the BOD₅. Again, more detailed investigation is necessary to allow any conclusion on the growth of the biomass in the reactors. Bacterial growth needs at least organic  carbon, a nitrogen source, phosphorus and some important trace metals. No further information is provided in the material and method section about the N and P source. Therefore, it remains unclear whether the growth rate might be limited with 10 % v/v diesel inoculum.

Ans.In this case, the diesel concentration is 5% with respect to the total work volume, and it is constant, what varies is the concentration of the inoculum of the microbial consortium and the growth rate is measured as an indicator that the bacteria are consuming the diesel. and therefore they are degrading it.
The composition of Davis' minimal medium was placed, this medium contains the required source of nitrogen and phosphorus.

However, it might also be that with higher concentration toxic effect of diesel or microbial competition for carbon decrease the growth rate.

Ans.Effectively. For this reason, one wants to find the optimal concentration of inoculum to avoid microbial competition due to quorum sensing when it is high, or very slow degradation if the inoculum is insufficient.

The authors are requested to clearly state the composition of the influence medium and loading rates. 

Anns. Culture medium concentration was placed, loading rates have not been calculated, but this will be taken into account for further investigation.

3. The removal of diesel was only measured at the end of the batch tests. Therefore, it remains in doubts how to interpret data obtained from BOD₅measurements only. The limited BOD₅ for 2% v/v diesel inoculum can be either a result of carbon limitation or due to other not investigated reasons. However, carbon limitation might a very expectable reason.

Ans. In this case there is no carbon limitation, diesel 2 is the carbon source.

4. The determination of the growth rates should be presented at least in a supplement file. The authors stated that they have performed all experiments in triplicate. Thus, the growth rate can be also reported with a standard deviation (SD). This is also true for the removal efficiency. 

Ans. Ok

5. I did not understand the requirement to perform statistical analyses such as ANOVA and Duncan’s test. It only demonstrates that the removal efficiencies are true. In fact, a similar outcome would be simply obtained using SD.

Ans. We decided to use the analysis of variance, so the output of the program that is presented in tables 3 and 4 provides us with additional important information that gives us greater support for the conclusions obtained after the investigation, the output of the sum of squares column provides us with the variation that exists between the groups (treatments) and within the groups (between repetitions), where a greater variation between the groups is obtained, which can be attributed to the effect of the independent variable, compared to the variation within groups that may be due to error between multiple measurements. In turn, it presents us with the output of the F statistic, where the F-calculated must be greater than the F-table to validate these differences. Finally, the value of the probability is presented in the sixth column (Table 02), which is tested with a significance of 5%, where it can be seen that this value is lower (0.00).
While in the DUNCAN post hoc tests (Table 03), they are relevant because they have allowed us to identify which groups are the same and which groups are different, validated by means of contrast statistics using a significance value of 5% to group equal means and means different.

6. Is there any reason to provide four digits (66.6667)? What is the meaning? Does it make your determined results more precise? 

Ans. It was considered to correct the figures leaving only 2 decimal places.

7. Result interpretation in the discussion occurs very theoretical due to missing

measurements.

Ans. I hope that with the new corrections your opinion can change.

Reviewer 3 Report

Dear authors,
I read your paper entitled "Effect of inoculum concentration on the degradation of diesel 2 by the Biot.PD001 consortium”.  The authors presented and explained the advantages of bioremediation procedures and the importance of of inoculum concentration on the degradation of Diesel 2 by the microbial consortium BIOT.PD001.
I believe that the paper is well written.
However, certain points need to be addressed for the manuscript to reach a level fit for publication in  Sustainability:
1.    Please use italics when writing bacterial names (Micrococcus, Pseudomonas etc.).
2.    Describe the microbial consortium in a little more detail, for example, which microorganisms are included in the consortium. If it is already a known consortium, give a reference to literature.
3.    Update the reference list a little bit by adding articles from 2020-2022

Author Response

Thank you very much dear colleague, for the comments provided. I send the answers to each comment.
best regards

_______________________________________________________________

1.    Please use italics when writing bacterial names (Micrococcus, Pseudomonas etc.). ____Ans: Scientific names were written in italics
2.     Describe the microbial consortium in a little more detail, for example, which microorganisms are included in the consortium. If it is already a known consortium, give a reference to literature._____Ans. The consortium was described
   3.    Update the reference list a little bit by adding articles from 2020-2022. Ans.__________References have been updated.

Reviewer 4 Report

This study evaluates the effect of the inoculum concentration on the degradation of Diesel 2 oil by the microbial consortium BIOT.PD001, under laboratory conditions; postulating the hypothesis that the effect of the concentration of inoculum on the degradation of Diesel 2 oil will be to increase the efficiency of the bioprocess in a directly proportional way.

It is undoubtedly an interesting and novel, however, it has many details that must be addressed in general, the authors must carefully study the manuscript and homogenize the text, in the attached document I include specific comments.

Comments for author File: Comments.pdf

Author Response

Thank you very much dear colleague, for the comments provided. I send the answers to each comment.
best regards

_______________________________________________________________

Ans. This study evaluates the effect of the inoculum concentration on the degradation of Diesel 2 oil by the BIOT.PD001 microbial consortium, under laboratory conditions; postulating the hypothesis that the effect of the inoculum concentration on the degradation of Diesel 2 oil will increase the efficiency of the bioprocess in a directly proportional way.
It is undoubtedly an interesting and novel, however, it has many details that must be addressed in general, the authors must carefully study the manuscript and standardize the text, in the attached document I include specific comments.

Introduction
1. The authors indicate an increase between 2000 and 2013 due to the growth of thermoelectric generation: what happened in the period between 2013 and 2022? two.
Ans.There are no data on diesel consumption in the years 2013 to 2022
2. In lines 38-62, it would be important to mention more recent data, or complete with more current information.
Ans.References have been updated.
3. line 99, you must use the correct form of references requested by the authors' manual of this journal to refer to "Ahmad (2017)".
Ans.Fixed citation style
4. It is recommended at the end of the introduction and within the abstract to add the novelty of the research.
Ans. Added the news of the study

Materials and methods
1. Establish where the study was developed and during what frequency.
Ans.It is indicated that the retention time was 14 days
2. It is recommended to add a graphic representation or photograph of the experimental phase.
Ans.Bioreactor design scheme was added and the process was better explained

Results and discussions
1. Figure 1 has information in Spanish, please change to English, in this same figure it is recommended to eliminate the internal lines of it.
Ans.Everything was written in English
2. Figures 2, 3 and 4 have information in Spanish, please change to English
Ans.Same
3. For a better understanding of the results obtained, it is recommended that the discussion section be included and discussed simultaneously in the presentation of results and not in a different section.
Ans.According to the structure of the article, the discussion goes after the results.
4. Figures 3 and 4 should be improved to clearly not see the numerical reference data well. conclusion
1. I consider it important to integrate the direction of future studies in this section.
General recommendations
 1. Review the correct way of referencing in the text, since it is not the one requested by the journal's author guide.

Ans.ok
2. Check the references section.

Ans.ok
3. Some paragraphs are too long, please check the English with a native speaker

Ans. ok.

4. Homogenize references and compound names throughout the document.

Ans. ok

Reviewer 5 Report

In this article, the authors try to determine the effect of inoculum concentration on the degradation of Diesel 2 by the microbial consortium isolated from active sludge from a Water Treatment Plant.

The results are interesting and these show that  the efficiency of the process increases as the concentration  of inoculum increases, obtaining the highest percentage of efficiency (94.77%) when using 10% of 25 inoculum (v/v).

The conclusions are relevant and drawn from the study.

The authors may refer to the following suggestions:

1.      Why did you use the name “Biot.PD001 consortium” in title of article and in all of article?  It’s a name consecrated for the microbial consortium  of bacteria of the genus  Bacillus,  Micrococcus, Pseudomonas and Rhodopseudomonas. I didn’t find it.  If not, I recommend change the name “Biot.PD001 consortium” and use for example: “bacteria consortium”.

2.      At Section Materials and methods you must present more details  about the experiments.

You must explain  the parameters from table 2, 3, 4.

3.      At Section Result, at table 4, Fig. 1, 2, 3, 4 you must use only English -not Spanish.

Author Response

Thank you very much dear colleague, for the comments provided. I send the answers to each comment.
best regards

_______________________________________________________________

1. Why did you use the name “Biot.PD001 consortium” in title of article and in all of article?  It’s a name consecrated for the microbial consortium  of bacteria of the genus  Bacillus,  Micrococcus, Pseudomonas and Rhodopseudomonas. I didn’t find it.  If not, I recommend change the name “Biot.PD001 consortium” and use for example: “bacteria consortium”.                    Ans. BIOT.PD001 is an internal code that is given in the Biotechnology Laboratory to new cultures that enter the collection.
2. At Section Materials and methods you must present more details  about the experiments.

You must explain  the parameters from table 2, 3, 4.

Ans.  The experiment was written in more detail.

3. At Section Result, at table 4, Fig. 1, 2, 3, 4 you must use only English -not Spanish.

Ans. language corrected

Round 2

Reviewer 2 Report

Standard deviation must be included in Table 1. There are several tipping mistakes to be corrected carefully before full acceptance.

Author Response

Dear colleague, thank you very much for your comments; Suggested changes to the manuscript were made.

Standard deviation must be included in Table 1.

Ans.Standard deviations were included in Table 1.

There are several tipping mistakes to be corrected carefully before full acceptance.

Ans. The document was revised and typing errors were corrected.
best regards

Author Response File: Author Response.pdf

Reviewer 4 Report

the authors attended my comments, I consider it appropriate to accept it

Author Response

Dear colleague, thank you very much for accepting the manuscript.
best regards

Reviewer 5 Report

It's OK.

Author Response

Dear colleague, thank you very much for accepting the manuscript.
best regards