Biodegradation of Malathion in Amended Soil by Indigenous Novel Bacterial Consortia and Analysis of Degradation Pathway
Round 1
Reviewer 1 Report
The manuscript describes “the biodegradation of malathion in amended soil by different indigenous novel bacterial consortia”. The theme is helpful for degradation and detoxification of environments contaminated with organophosphate.
The main revision suggestion are as follows.
In materials and methods, the design of the experiment and some data are suggested to be supplemented. For example, the density of inoculum, the soil culture condition (soil moisture, temperature, etc.), The detect time for Metabolites analysis.
In “3.3. Metabolites analysis”, it is suggested to written clearly that the metabolites were from which bacterial consortia biodegradation process.
Some sentences had grammar errors. For example, “The pure bac-9 terial strains efficiently degraded 50.16-68.47% of pesticide within 15 days of incubation, and me-10 tabolites were observed to accumulate in the soil. “
Author Response
(Manuscript ID: soilsystems-2540554)
Thank you very much for taking the time to review this manuscript. Please find the detailed responses below and the corresponding revisions/corrections highlighted/in track changes in the re-submitted files.
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Reviewer - 1: Comments |
The manuscript describes “the biodegradation of malathion in amended soil by different indigenous novel bacterial consortia”. The theme is helpful for degradation and detoxification of environments contaminated with organophosphate.
The modifications/changes have been highlighted in green in the revised manuscript.
Comment 1 In materials and methods, the design of the experiment and some data are suggested to be supplemented. For example, the density of inoculum, the soil culture condition (soil moisture, temperature, etc.), The detect time for metabolites analysis
Response 1 As suggested by the reviewer, the experimental design section was checked and the said suggestions are already supplemented, highlighted in green in the revised manuscript. Metabolite analysis time is mentioned in result and discussion section i.e., 15th day.
Comment 2 In “3.3. Metabolites analysis”, it is suggested to written clearly that the metabolites were from which bacterial consortia biodegradation process.
Response 2 The suggested amendments were made in this revision
Comment 3 Some sentences had grammar errors. For example, “The pure bac-9 terial strains efficiently degraded 50.16-68.47% of pesticide within 15 days of incubation, and me-10 tabolites were observed to accumulate in the soil.
Response 3 We have tried our best to improve the English and remove grammatical errors as suggested by the reviewer. However, at some places as highlighted by the reviewer, these errors are due to page numbers/manuscript formatting done by the journal, and were asked do not modify the same.
Reviewer 2 Report
The manuscript "Biodegradation of malathion in amended soil by indigenous novel bacterial consortia and analysis of degradation pathway" is a paper describing the ability of three bacterial strains to degrade malathion and the possible degradation pathway. The paper is relevant, but there are some points that should be checked which I mention below:
Major comments:
The identification of the isolates included in this work is missing. The authors mention that this identification has been done in previous works (references 6 and 24); however, in reference 24 they only identify and evaluate the degradation capacity by the Bacillus sp. AGM5 and reference 6 corresponds to a review of the state of the art on the persistence of phorate. Therefore, the other isolates (AGB3 and MAGK3) have not been identified and have not been reported either in the work of reference 24 or in the present one. Furthermore, the doubt remains because the strain AGM5 was named as Bacillus sp. AGM5 in the previous paper (Reference 24) and Bacillus paramycoides AGM5 in the present paper. Why was the strain re-named? Please clarify this change.
The results of the degradation kinetics of malathion by the individual strains or by the consortium described in Figure 1 should be reported in the Results section and not in the Materials and Methods section. Materials and methods should only describe how the experiment was set up, the number of replicates used, the number of terminal samples included, the days of sampling, the controls included and the purpose of their inclusion. In addition, the quality of the plots in Figure 1 should be improved and transferred to the results. The three plots need to be homogenised, including the plotted points, i.e. the same sampling days need to be plotted. In b) what happened to the last two points corresponding to the consortium (three strains) and B. cereus + B. paramycoides?
Specify in the text that the uninoculated unsterilized control is used to evaluate photolysis, adsorption phenomena and the involvement of the native soil-associated microbiota, and that the uninoculated sterilized control is used to evaluate adsorption phenomena, and that possibly in this study the microbiota that achieved survive a single round of sterilization. From this last point, why did the authors include only a single sterilization cycle instead of at least three sterilization cycles with recovery intervals between each of them?
Regarding the degradation kinetics of the compound, were the sampling points taken from terminal samples or were they taken from the same vial or microcosm? If it was the latter, it is possible that there is a bias since the microcosm lost sample during the kinetics (10.5 gr at each sampling point) and therefore, the microcosm varies between each sampling point and no longer has the same conditions considered at the beginning of the experiment.
In addition, the evaluation of microbial growth during the degradation kinetics of the compound was not included, which could be done through the evaluation of CO2.
It remains to be discussed that the participation of the microbiota associated with the soil, which is very significant since in the control of non-sterile soil an elimination of the compound of 81.887% was obtained and at 15 days of 34.452%; that is, the 100% removal value obtained by the consortium at 15 days, 34.452% corresponds to the participation of the microbiota associated with the soil and other abiotic processes. Although a soil with no history of contamination with the compound under study was used, the soil-associated microorganisms have the potential to degrade it. In addition, it should be emphasized that other non-abiotic processes, such as adsorption or photocatalysis, are also involved in the degradation of the compound, since a reduction of 52,458% was achieved after 30 days. These points need to be discussed in detail.
In terms of the proposed metabolic pathway, to whom is the proposed degradation pathway attributed? To which isolate, to the consortium, or to the native soil microbiota? please clarify and discuss this.
Minor comments:
Change “environs” to “environment” on line 29, 48 and 71.
Add a comma between “vegetables” and “etc.” on line 36.
Write microorganisms in italics, on line 101 and 102.
Improve the quality of figures and graphs.
no comments
Author Response
Reviewers - 2: Comments
The manuscript "Biodegradation of malathion in amended soil by indigenous novel bacterial consortia and analysis of degradation pathway" is a paper describing the ability of three bacterial strains to degrade malathion and the possible degradation pathway. The paper is relevant, but there are some points that should be checked which I mention below:
Comment 1 The identification of the isolates included in this work is missing. The authors mention that this identification has been done in previous works (references 6 and 24); however, in reference 24 they only identify and evaluate the degradation capacity by the Bacillus sp. AGM5 and reference 6 correspond to a review of the state of the art on the persistence of phorate. Therefore, the other isolates (AGB3 and MAGK3) have not been identified and have not been reported either in the work of reference 24 or in the present one. Furthermore, the doubt remains because the strain AGM5 was named as Bacillus sp. AGM5 in the previous paper (Reference 24) and Bacillus paramycoides AGM5 in the present paper. Why was the strain re-named? Please clarify this change.
Response 1 Among the three strains used in this study, two were previously identified and reported in references 24, and 45. However, third strain Bacillus cereus (AGB3) has been communicated in another journal and is under review and its accession number has been reported in that manuscript.
AGB3 is Bacillus cereus while MAGK3 is Bacillus paramycoides, two different strains of the same genera.
Comment 2 The results of the degradation kinetics of malathion by the individual strains or by the consortium described in Figure 1 should be reported in the Results section and not in the Materials and Methods section. Materials and methods should only describe how the experiment was set up, the number of replicates used, the number of terminal samples included, the days of sampling, the controls included and the purpose of their inclusion.
Response 2 As suggested by the reviewer the results of the degradation kinetics described in Fig. 1 has been moved to results and discussion section from materials and methods section
Comment 3In addition, the quality of the plots in Figure 1 should be improved and transferred to the results. The three plots need to be homogenized, including the plotted points, i.e. the same sampling days need to be plotted. In b) what happened to the last two points corresponding to the consortium (three strains) and B. cereus + B. paramycoides?
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We have tried our best to improve the quality of the plots in Fig. 1 as suggested by the reviewer. Different plotting points were placed to differentiate between the different treatments. |
Response 3 The last two points corresponding to the consortium are missing due to the complete disappearance of malathion within 21 days in all the consortia of different bacterial combinations.
Comment 4 Specify in the text that the uninoculated unsterilized control is used to evaluate photolysis, adsorption phenomena, and the involvement of the native soil-associated microbiota, and that the uninoculated sterilized control is used to evaluate adsorption phenomena, and that possibly in this study the microbiota that achieved survive a single round of sterilization. From this last point, why did the authors include only a single sterilization cycle instead of at least three sterilization cycles with recovery intervals between each of them?
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Response 4 The suggestions of reviewer have been included and highlighted in green in the revised manuscript. 3 successive sterilizations at 121 °C, at 24 h intervals for 20 min were used in this study which has been mentioned in the revised manuscript as suggested by the reviewer.
Comment 5 Regarding the degradation kinetics of the compound, were the sampling points taken from terminal samples or were they taken from the same vial or microcosm? If it was the latter, it is possible that there is a bias since the microcosm lost sample during the kinetics (10.5 gr at each sampling point) and therefore, the microcosm varies between each sampling point and no longer has the same conditions considered at the beginning of the experiment.
Response 6 As the experiments were conducted in complete randomized block design in a pot culture experiment (Plastic cups), considering all other parameters in control except degradations kinetics. For any sampling this much variation is expected as suggested by reviewer, however experiments done in triplicates and hence overrule any such discrepancies.
Comment 7 In addition, the evaluation of microbial growth during the degradation kinetics of the compound was not included, which could be done through the evaluation of CO2.
Response 7 This good point has been suggested by reviewer , and can be considered in our future research however we were relying more on degradation parameters as an outcome of microbial activity based on fundamental flask lab scale experiments
Comment 8 It remains to be discussed that the participation of the microbiota associated with the soil, which is very significant since in the control of non-sterile soil an elimination of the compound of 81.887% was obtained and at 15 days of 34.452%; that is, the 100% removal value obtained by the consortium at 15 days, 34.452% corresponds to the participation of the microbiota associated with the soil and other abiotic processes. Although a soil with no history of contamination with the compound under study was used, the soil-associated microorganisms have the potential to degrade it. In addition, it should be emphasized that other non-abiotic processes, such as adsorption or photocatalysis, are also involved in the degradation of the compound, since a reduction of 52,458% was achieved after 30 days. These points need to be discussed in detail.
Response8 The suggestions of the reviewer have been included in the revised manuscript
Comment 9 In terms of the proposed metabolic pathway, to whom is the proposed degradation pathway attributed? To which isolate, to the consortium, or to the native soil microbiota? please clarify and discuss this.
Response9 The proposed metabolic pathway was attributed to the consortium (consisting of all three strains), as it shows the optimum results as compared to other treatments.
Comment 10 Minor comments: Change “environs” to “environment” on line 29, 48 and 71. Add a comma between “vegetables” and “etc.” on line 36. Write microorganisms in italics, on line 101 and 102.Improve the quality of figures and graphs
Response10 The corrections/modifications have been done as suggested by the reviewer.
Reviewer 3 Report
Please see the attachment
Comments for author File: 
Comments.pdf
The English language is good enough to understand.
Author Response
Reviewers - 3: Comments
Comment 1 The manuscript entitled "Biodegradation of malathion in amended soil by indigenous novel bacterial consortia and analysis of degradation pathway” by Mohd Ashraf Dar and Garima Kaushik described their research findings on the degradation of malathion using three bacteria combinedly and single. The consortia of three bacteria could degrade malathion more effectively, and complete malathion removal was observed by the 15th day. In general, the manuscript is written well.
Response1 The authors are very thankful to the reviewer for his encouraging comments.
Comment 2 The authors claimed this is the FIRST REPORT to use a bacterial consortium for successful malathion degradation. This statement is not correct. The authors should reframe their statement. Many studies have already been published for malathion degradation using either a single or a consortium of bacteria. I think this manuscript is unsuitable for publication in this version.
Response2 As suggested by the reviewer, it is true there are many studies that reported malathion degradation by single as well as by consortium. They are true in the case of aqueous cultures, but we have demonstrated the degradation mechanism of malathion by different bacterial consortia in soil for the first time especially with these new strains
Comment3 The introduction is written well.
Response3 Authors are very thankful to the reviewer for his encouraging comment.
Comment4 Line 96, the authors could include the temperature along with pressure.
Response4 The reviewer’s suggestion has been included in the revised manuscript.
Comment5 Line 101-102, the Bacterial name should be in italic.
Response5 As suggested by the reviewer, we have cross-checked the same and corrected in the revised manuscript
Comment6 Line 129, What is the rationale for using 500 mgkg-1 concentration?
Response6 500 mgkg-1 concentration of malathion was used in this study because at this concentration all three bacterial strains showed the optimum results in aqueous cultures
Comment7 Line 102-103, ……..performed as described previously in our published reports (6, 24).
Response7 The sentence has been corrected as well as the citation
Comment8 Line 105, ………….the soil for the first time. This shatement shoulb be changed.
Response8 As suggested by the reviewer the statement has been changed
Comment9 Line 115, washed with sterilized N-saline? Does it mean NaCl saline?
Response9 Yes, it is NaCl saline and has been corrected in the revised manuscript as suggested.
Comment10 Line 134-138, How the soil samples were taken for the analysis? Though it is mentioned in section 2,6, the soil sampling should be precise for the analysis.
Response10 Representative samples were aseptically taken at different time intervals and were extracted using the QuEChERS method as described in 2.6 section.
Comment11 Line 142, the equation is in the bigger front.
Response11 The font of the equation has been reduced as suggested by the reviewer.
Comment12 Line 146-148, 10g of soil were transferred to screwed glass tubes followed by the addition of 5 ml ultrapure water. How is it possible to take 10 ml supernatant after vortex and centrifugation?
Response12 It was actually the volume of extraction solvent that was mistakenly mentioned as supernatant and has been corrected in the revised manuscript as suggested by the reviewer.
Comment13 Section 2,8, need to rewrite as it is copied from other source
Response13 We tried our best to rewrite the section as suggested by the reviewer
Comment14
Line 248-251, malathion degradations were 81% and 52% in control samples. What could be the reasons other than microbes were present in the soil? A possible explanation can be done as follows: many environmental factors such as physical and nutritional conditions for bacterial growth, interaction with other microbes existing in nature, and presence of other chemical substances in soils that may interact for the degradation…………..
Response14 As suggested by the reviewer, the other possible factors responsible for the degradation have been mentioned in the revised paper as suggested by the reviewer.
Comment15 Figure 2 should be more clear
Response15 The clarity of Fig. 2 has been improved in the revised manuscript as suggested.
Reviewer 4 Report
Manuscript Title: Biodegradation of malathion in amended soil by indigenous novel bacterial consortia and analysis of degradation pathway
Authors: Mohd Ashraf Dar , Garima Kaushik
The work provides some interesting results concerning the microbial degradation of malathion in soil by indigenous bacterial consortia. Generally speaking, this paper was well organized and the data was properly testified. However, the following suggestions should be considered.
Major comments:
There are many reports on the use of strains to degrade malathion. It is suggested to add a comparison with the existing reported results of bacterial degradation of malathion at the end of the paper to clarify the innovation of this study.
Technical queries/ suggestions:
1. Abstract should be written more precisely and explain novelty of this work. Microbial degradation of malathion has been widely reported, so what is the novelty of this work? Please briefly mention the main aim of the work and highlight the principal conclusions. In addition, the novelty and significance of the manuscript were not highlighted in this section, please modify the Abstract more clearly.
2. Introduction should be improved. Introduction should briefly place the study in a broad context and highlight why it is important. It should define the purpose of the work and its significance. The current state of the research field should be reviewed, and key publications cited. Finally, briefly mention the main aim of the work and highlight the principal conclusions. However, the novelty and significance of the manuscript were not highlighted in the Introduction section, please modify the introduction more clearly.
3. Lines 45-57: Please highlight the specific effects of malathion pollution. What toxic effects of malathion have on soil, plant, animal, and humans? How fast the malathion metabolize in the environment? I think it is favorable to show its half-life in this part based on the literature available and described in original paper. This way the authors will demonstrate that they really have a good knowledge of the related literature.
4. Lines 58-62: What are the advantages of biodegradation/biotransformation as compare to the physicochemical methods?
5. Lines 75-77: As stated by the authors, Bacillus strains have been reported to degrade malathion. So what is the novelty of this work? Why the authors select the Bacillus strains for study?
6. Lines 82-84: Have the described microbial strains been deposited in a public strain collection? Please mention the collection number in the manuscript.
7. Lines 75-85: Please check all the species names. Species names are typically given in full the first time they are used within the main text and then abbreviated throughout the remainder of the text.
8. Line 101-102: Check the species names.
9. Line 156: UHPLC, GC-MS, please use full names here. Please check throughout the manuscript that abbreviations/acronyms are defined the first time they appear in each of three sections: the abstract; the main text; the first figure or table.
10. Line 189: Move Fig.1 to the Results and discussion section. This figure is not clear. The quality should be significantly improved.
11. Statistical analysis is very important. I suggest the authors add a new section in the Materials and Methods to describe the details of the statistical analysis.
12. Results and discussion. The poor discussion of the results. Author have showed the great amount of results that they have achieved, but they did not use them to develop an interesting discussion which could supplement to earlier studies on biodegradation processes carried out by pure or mixed cultures. Bacillus strains are known for their metabolic capability and environmental versatility as well as for their ability to manage bacterial and fungal pathogens infecting crop plants. Authors should add more information into this section and cite the recent research into the field. However, the authors did not compare the degradation activity of the selected Bacillus strains with that of other Bacillus strains based on the literature such as Mechanism of β-cypermethrin metabolism by Bacillus cereus GW-01. Chem. Eng. J. 430, 2022; New role for a commercially available bioinsecticide: Bacillus thuringiensis Berliner biodegrades the pyrethroid cypermethrin. Environ. Sci. Technol., 2021; Novel mechanism and degradation kinetics of allethrin using Bacillus megaterium strain HLJ7 in contaminated soil/water environments. Environ. Res. 2022.
13. Line 340: Figure 3 is not clear. The quality should be significantly improved.
14. Conclusion: This section should be revised for the better understanding of the topic and its future research.
15. References: Many of the references have been superceded and more modern ones are required.
Author Response
Reviewers - 4: Comments
Comment1 Major comments: There are many reports on the use of strains to degrade malathion. It is suggested to add a comparison with the existing reported results of bacterial degradation of malathion at the end of the paper to clarify the innovation of this study
Response1 As suggested by the reviewer, the comparison with the existing reported results of bacterial degradation of malathion is reported in 3.2 section of result and discussion.
Comment2 Abstract should be written more precisely and explain novelty of this work. Microbial degradation of malathion has been widely reported, so what is the novelty of this work? Please briefly mention the main aim of the work and highlight the principal conclusions. In addition, the novelty and significance of the manuscript were not highlighted in this section, please modify the Abstract more clearly.
Response2 The suggestions of the reviewer have been included in the revised manuscript
Comment3Introduction should be improved. Introduction should briefly place the study in a broad context and highlight why it is important. It should define the purpose of the work and its significance. The current state of the research field should be reviewed, and key publications cited. Finally, briefly mention the main aim of the work and highlight the principal conclusions. However, the novelty and significance of the manuscript were not highlighted in the Introduction section, please modify the introduction more clearly
Response3 The suggestions of the reviewer have been included in the revised manuscript
Comment4 Lines 45-57: Please highlight the specific effects of malathion pollution. What toxic effects of malathion have on soil, plant, animal, and humans? How fast the malathion metabolize in the environment? I think it is favorable to show its half-life in this part based on the literature available and described in original paper. This way the authors will demonstrate that they really have a good knowledge of the related literature.
Response4 The suggestions has been included in the revised manuscript as suggested by the reviewer
Comment5 Lines 58-62: What are the advantages of biodegradation/biotransformation as compare to the physicochemical methods?
Response5 The suggestion has been included in the revised manuscript as suggested by the reviewer.
Comment6 Lines 75-77: As stated by the authors, Bacillus strains have been reported to degrade malathion. So what is the novelty of this work? Why the authors select the Bacillus strains for study?
Response6 Research on malathion biodegradation was mostly carried out by single strains including Bacillus species. Hence, it is important to establish a biosystem (consortia) that can efficiently degrade such pesticides. Because mixed cultures of microbial consortium typically displayed improved productivity and substrate tolerance compared to pure cultures of a single strain. Therefore, the present study was intended to develop a highly efficient bacterial consortia to evaluate the bioremediation of malathion in soil by this bacterial consortia.
Comment7 Lines 82-84: Have the described microbial strains been deposited in a public strain collection? Please mention the collection number in the manuscript.
Response7 The sequences of strains were submitted to NCBI database and their submission to a public strain collection is under process.
Comment8 Lines 75-85: Please check all the species names. Species names are typically given in full the first time they are used within the main text and then abbreviated throughout the remainder of the text.
Response8 The same has been checked and corrected as suggested by the reviewer.
Comment9 Line 101-102: Check the species names
Response9 The species names have been checked throughout the manuscript as suggested.
Comment10 Line 156: UHPLC, GC-MS, please use full names here. Please check throughout the manuscript that abbreviations/acronyms are defined the first time they appear in each of three sections: the abstract; the main text; the first figure or table.
Response10 The suggestions of the reviewer have been included in the revised manuscript
Comment11 Line 189: Move Fig.1 to the Results and Discussion section. This figure is not clear. The quality should be significantly improved.
Response11 Fig. 1 has been moved to the results and discussion section and its quality has been improved too as suggested by the reviewer.
Comment12 Statistical analysis is very important. I suggest the authors add a new section in the Materials and Methods to describe the details of the statistical analysis
Response12 Thank you for the suggestions, we will try to keep different section on statistical analysis in our next manuscript, However in this manuscript also we did perform all statistical analysis and considered in result section.
Comment13 Results and discussion. The poor discussion of the results. Author have showed the great amount of results that they have achieved, but they did not use them to develop an interesting discussion which could supplement to earlier studies on biodegradation processes carried out by pure or mixed cultures. Bacillus strains are known for their metabolic capability and environmental versatility as well as for their ability to manage bacterial and fungal pathogens infecting crop plants. Authors should add more information into this section and cite the recent research into the field. However, the authors did not compare the degradation activity of the selected Bacillus strains with that of other Bacillus strains based on the literature such as Mechanism of β-cypermethrin metabolism by Bacillus cereus GW-01. Chem. Eng. J. 430, 2022; New role for a commercially available bioinsecticide: Bacillus thuringiensis Berliner biodegrades the pyrethroid cypermethrin. Environ. Sci. Technol., 2021; Novel mechanism and degradation kinetics of allethrin using Bacillus megaterium strain HLJ7 in contaminated soil/water environments. Environ. Res. 2022.
Response13 The suggested references have been added in the revised manuscript.
Comment14 Line 340: Figure 3 is not clear. The quality should be significantly improved.
Response14 The quality of Fig. has been improved in the revised manuscript as suggested by the reviewer.
Comment15 Conclusion: This section should be revised for the better understanding of the topic and its future research.
Response15 The conclusion has been modified as suggested
Comment16 References: Many of the references have been superseded and more modern ones are required.
Response16 More latest references have been added as suggested by the reviewer
Reviewer 5 Report
The manuscript by Dar and Kaushik described bioaugmentation for enhancing malathion degradation using microbial consortia containing Micrococcus and Bacillus spices. Such microbial consortia may be useful for bioremediation of contaminated environment. Overall, the study is well designed, and the text and data presentation are also clear. However, there are several parts that may need additional information and explanations for clarification. Please consider the following for further improvement.
1. The Introduction lacks information about the biodegradation pathway of malathion. Can microbes completely degrade/mineralize malathion? Are there intermediate metabolites produced/accumulated that are still harmful to the environment?
2. Line11, 23. Better to use “bacterial spices”, rather than bacterial sp/spp (never saw this before).
3. Line86-87. Why using consortia is better than using pure cultures? Need more explanation here.
4. Line100-103. Not clear. Were the enrichment and isolation performed in this study? If not, just state that these bacterial strains were obtained from earlier studies.
5. Line108-109. Should be 16S rRNA gene.
6. Line116 and line133. What is the cell amount or biomass in the 2% of cell suspensions and 2 ml overnight cultures? I assume the initial biomass inoculated was technically the same in the cultures with individual strains or consortia. Otherwise, the conclusion of this study might be problematic. As the observed different malathion degradation rate by the cultures may simply because of the different biomass inoculated at the beginning.
7. Fig1. The LogC values for B. cereus + B. paramycoides and M. aloeverae + B. cereus + B. paramycoides in graph (b) are missing at day-20. Undetectable values should also be indicated/reflected in the graphs. Moreover, it’s better to use the same vertical and horizontal scales (LogC and Time) for the three graphs. So the readers can compare the degradation rate/kinetics of different conditions more easily.
8. Line212. What do you mean by “The growth of M. aloeverae, B. cereus and B. paramycoides individually in inoculated cultures”? Better to re-phrase. It also needs to be noted that there is no data in this study shows these strains can grow with malathion.
9. The unit of malathion concentration in the text and Tables (1,2,3) is confused. i.e., sometimes mg/kg, sometimes μL/kg.
10. Line252. According to Table 3 the degradation rate is 52.458%, not 48%.
11. Figure 2. So these degradation products were detected in which cultures? Cultures with individual bacterial strains? Or with mixed strains? Or control cultures with only soils? Do you expect different degradation products/pathways under the different conditions? Can you extend the discussion regarding this?
12. Same for Figure 3. Degradation pathway of which condition? It shows trimethyl thiophosphate is the end product for malathion degradation. Is trimethyl thiophosphate still harmful for the environment? Can this compound be further degraded or mineralized by microbes in the environment? Need to discuss a little bit more here.
Author Response
Reviewers - 5: Comments
The manuscript by Dar and Kaushik described bioaugmentation for enhancing malathion degradation using microbial consortia containing Micrococcus and Bacillus spices. Such microbial consortia may be useful for bioremediation of contaminated environment. Overall, the study is well designed, and the text and data presentation are also clear. However, there are several parts that may need additional information and explanations for clarification. Please consider the following for further improvement.
Comment1 The Introduction lacks information about the biodegradation pathway of malathion. Can microbes completely degrade/mineralize malathion? Are there intermediate metabolites produced/accumulated that are still harmful to the environment?
Response1 The intermediate metabolites are also harmful to the environment but as shown in the pathway these metabolites are further degraded into thiophosphates, which are less toxic as compared to malathion and other metabolites.
Comment2 Line11, 23. Better to use “bacterial spices”, rather than bacterial sp/spp (never saw this before).
Response2 Sp/spp. has been removed by species as suggested by the reviewer
Comment3 Line86-87. Why using consortia is better than using pure cultures? Need more explanation here
Response3 The explanation has been included in the revised manuscript as suggested by the reviewer.
Comment4. Line100-103. Not clear. Were the enrichment and isolation performed in this study? If not, just state that these bacterial strains were obtained from earlier studies
Response4 As suggested by the reviewer, the statement has been modified to make it clearer
Comment5 Line108-109. Should be 16S rRNA gene.
Response5 The sentence has been corrected as suggested by the reviewer.
Comment6 Line116 and line133. What is the cell amount or biomass in the 2% of cell suspensions and 2 ml overnight cultures? I assume the initial biomass inoculated was technically the same in the cultures with individual strains or consortia. Otherwise, the conclusion of this study might be problematic. As the observed different malathion degradation rate by the cultures may simply because of the different biomass inoculated at the beginning.
Response6 All inoculations were made at 2% i.e., the equivalent of 2 ml inoculum to 100 g soil. The inoculation was the same in the cultures of individual strains as well as in consortia
Comment7 Fig1. The LogC values for B. cereus + B. paramycoides and M. aloeverae + B. cereus + B. paramycoides in graph (b) are missing at day-20. Undetectable values should also be indicated/reflected in the graphs. Moreover, it’s better to use the same vertical and horizontal scales (LogC and Time) for the three graphs. So the readers can compare the degradation rate/kinetics of different conditions more easily.
Response7 The LogC values for B. cereus + B. paramycoides and M. aloeverae + B. cereus + B. paramycoides in graph (b) are missing at day-20, because malathion was completely degraded by 20th day in these consortia combinations except in M. aloeverae and B. paramycoides consortium. The scales are the same in all the graphs, however, due to the degradation rate of different bacteria combinations of consortia, some changes have occurred.
Comment8 Line212. What do you mean by “The growth of M. aloeverae, B. cereus and B. paramycoides individually in inoculated cultures”? Better to re-phrase. It also needs to be noted that there is no data in this study shows these strains can grow with malathion.
Response8As suggested by the reviewer, the sentence has been rephrased in the revised manuscript. As mentioned earlier, these strains were isolated during our previous studies, and their growth kinetics were also checked
Comment9 The unit of malathion concentration in the text and Tables (1,2,3) is confused. i.e., sometimes mg/kg, sometimes μL/kg.
Response9 The units have been uniformed throughout the manuscript as suggested by the reviewer.
Comment10. Line252. According to Table 3 the degradation rate is 52.458%, not 48%.
Response10 The same has been corrected in the revised manuscript as suggested by the reviewer.
Comment11 Figure 2. So these degradation products were detected in which cultures? Cultures with individual bacterial strains? Or with mixed strains? Or control cultures with only soils? Do you expect different degradation products/pathways under the different conditions? Can you extend the discussion regarding this?
Response11 As the optimum degradation results were shown by the consortium of all the three strains. So, metabolites formed during the degradation kinetics of this consortium were characterized and possible degradation pathway was proposed.
Comment12 Same for Figure 3. Degradation pathway of which condition? It shows trimethyl thiophosphate is the end product for malathion degradation. Is trimethyl thiophosphate still harmful for the environment? Can this compound be further degraded or mineralized by microbes in the environment? Need to discuss a little bit more here.
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Response12 As already discussed in section 3.3., this pathway is for the consortium of all three strains. |
The end product trimethyl thiosulphate metabolite is less toxic as compared to malathion and other metabolites. This compound can be further degraded into simpler phosphates as shown by the same strains in aqueous cultures (Ref- 24, 45).
Round 2
Reviewer 2 Report
The manuscript improved a lot, however the following points needed to be modified:
In order to be more precise in naming the three strains used in this study, their names must be cited appropriately in the manuscript, that is, writing genus, species, and strain. e.g. Micrococcus aloeverae MAGK3, Bacillus cereus AGB3 and Bacillus paramycoides AGM5. The above is important because surely not all members of the bacterial species used in this work have the capacity to degrade malathion.
Fig. 1 was included in the results section, but please change it to the section where this result was generated. It can be mentioned in section 3.2. "Bioremediation evaluation of individual strains and mixed strains consortium", second paragraph and include Fig. 1 near its mention.
Sterilization cycles should be specified as consisting of [121°C, 15 psi pressure, 20 minutes] with 24-h intervals between each cycle.
The answer to Comment 5 is not clear to me. Were terminal samples used or not?
In microcosms using soils, it is common to consider the total number of microcosms (problems and controls) in such a way that at least “n=3”, although there are several studies that use n=9. In fact, random and terminal samples are taken at each considered sampling point and with n=9 the possible variability is minimized.
Separate "500mgkg-1" to "500" insert space "mgKg-1"
Write in italics the names of the microorganisms, including the legend of Fig., the above applies to the legend of Fig. 1.
Author Response
Reviewers - 2: Comments
The manuscript improved a lot, however the following points needed to be modified
Comment 1: In order to be more precise in naming the three strains used in this study, their names must be cited appropriately in the manuscript, that is, writing genus, species, and strain. e.g. Micrococcus aloeverae MAGK3, Bacillus cereus AGB3 and Bacillus paramycoides AGM5. The above is important because surely not all members of the bacterial species used in this work have the capacity to degrade malathion.
Response 1: Thank you for your excellent suggestion. The names of strains used in the study were modified and cited accordingly as suggested by the reviewer.
Comment 2: Fig. 1 was included in the results section, but please change it to the section where this result was generated. It can be mentioned in section 3.2. "Bioremediation evaluation of individual strains and mixed strains consortium", second paragraph and include Fig. 1 near its mention.
Response 2: The results of the degradation kinetics described in Fig. 1 (renamed as Fig. 3 in revised manuscript as per another reviewer suggestion) were moved to section 3.4. “Kinetic studies” instead of section 3.2. because Fig. 3. shows the kinetics of malathion degradation by plotting malathion residues against time in amended soil, which is discussed in section 3.4.
Comment 3: Sterilization cycles should be specified as consisting of [121°C, 15 psi pressure, 20 minutes] with 24-h intervals between each cycle.
Response 3: The suggestions of the reviewer have been included in the revised manuscript.
Comment 4: The answer to Comment 5 is not clear to me. Were terminal samples used or not?
Response 4: Samples were taken from the same microcosm at different time intervals. As the experiments were conducted in complete randomized block design in a pot culture experiment (Plastic cups), considering all other parameters in control except degradations kinetics. For any sampling this much variation is expected as suggested by reviewer, however experiments done in triplicates and hence overrule any such discrepancies.
Comment 5: In microcosms using soils, it is common to consider the total number of microcosms (problems and controls) in such a way that at least “n=3”, although there are several studies that use n=9. In fact, random and terminal samples are taken at each considered sampling point and with n=9 the possible variability is minimized
Response 5: In our studies we have used n=3 i.e., experiments were performed in triplicates to reduce any error.
Comment 6: Separate "500mgkg-1" to "500" insert space "mgKg-1"
Response 6: The change/modification has been done in the revised manuscript as suggested by the reviewer
Comment 7: Write in italics the names of the microorganisms, including the legend of Fig., the above applies to the legend of Fig. 1
Response 7: The name of bacterial strains in Fig. legends as well as in the whole manuscript has been made italic in the revised manuscript as suggested by the reviewer.
Reviewer 3 Report
The authors have improved the manuscript according to the comments. I have few minor comments to address:
1. The authors mentioned in the abstract that “this is the first report to use a bacterial consortium for successful malathion degradation.” Actually, this is not the first report. There are many reports already have been published.
2. An explanation is added in lines 267-274. Please mention a reference here.
3. Please number the figures correctly and cite the figure in the text accordingly. For example, the last figure just above the Conclusion is Figure 1.
4. There are many typo mistakes. Eg. 500mgkg-1. keep space between number and mg/kg
English language is good enough for understanding
Author Response
Reviewers - 3: Comments
The authors have improved the manuscript according to the comments. I have few minor comments to address.
Comment 1: The authors mentioned in the abstract that “this is the first report to use a bacterial consortium for successful malathion degradation.” Actually, this is not the first report. There are many reports already have been published.
Response 1: As suggested by the reviewer, the sentence has been modified.
Comment 2: An explanation is added in lines 267-274. Please mention a reference here.
Response 2: As suggested by the reviewer, the reference has been added.
Comment 3: Please number the figures correctly and cite the figure in the text accordingly. For example, the last figure just above the Conclusion is Figure 1.
Response 3: The numbering as well as citation of figures in the text has been modified as suggested by the reviewer.
Comment 4: There are many typo mistakes. E.g. 500mgkg-1. keep space between number and mg/kg
Response 4: The reviewer’s suggestion has been included in the revised manuscript
Reviewer 4 Report
The authors have considered all comments raised by the reviewers and revised the manuscript accordingly based on these comments. The revision is fine and can be accepted for publication in current form.
Author Response
Reviewers - 4: Comments
Comment 1: The authors have considered all comments raised by the reviewers and revised the manuscript accordingly based on these comments. The revision is fine and can be accepted for publication in current form.
Response 1: The authors are very thankful to the reviewer for their encouraging comments.
