Environmentally Friendly and Cost-Effective Approaches to Reduce Toxin Content in Toxic Cyanobacterial Biomasses

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

Comments to the Authors

GENERAL COMMENTS

The manuscript titled: “Environment-friendly and cost-effective approaches to reduce toxin content in toxic cyanobacterial biomasses” - ID: biomass-2956833 is interesting.

I find the article intriguing because it addresses the pressing issue of cyanobacterial toxicity in aquatic ecosystems. With the expansion of Cyanobacteria Harmful Algal Blooms (CyanoHABs) due to climate change and anthropogenic pollution, there is a growing concern about the health risks posed by these blooms to both humans and the environment. The study's focus on reducing toxin levels in cyanobacteria biomass, particularly Microcystis aeruginosa and Chrysosporum ovalisporum strains, is particularly relevant given the potential for these organisms to produce potent toxins. By evaluating various treatments, including exposure to sunlight and UV radiation, the research offers valuable insights into effective strategies for mitigating cyanobacteria toxicity. This knowledge is crucial for developing environmentally friendly approaches to address the harmful effects of cyanobacterial blooms, ultimately contributing to the preservation of water quality and ecosystem health.

 

 

SPECIFIC COMMENTS

Abstract

The abstract is properly edited.

 

Introduction

The introduction is interesting and provides essential information regarding the significance of cyanobacteria. It may be worthwhile to delve further into the utilization of cyanobacteria in biofuel production and as fertilizers. This would underscore the importance of the conducted research, demonstrating the significance of cyanobacteria in the economy. I understand that a sentence on this topic is already included in the introduction, but it might be beneficial to add the latest statistical data on the use of cyanobacteria in tons and the quantity of biofuels produced from them. It would also be valuable to mention the leading countries in this field worldwide. I believe this is important, especially considering that the authors cite limited and outdated literature. Only 16 references pertain to studies from the last 10 years, including 6 from 2020 and above. This is a weakness of the manuscript.

page 2, line 86

The introduction is quite engaging; however, the final paragraph lacks a clearly stated research objective, and no research hypothesis is posited.

 

Materials and Methods

Chapter 2. Materials and Methods is well described. In subsection 2.5. Statistical Analysis, if the authors incorporate the suggestion for improving the graphs, I propose marking homogeneous groups for the mean bars and adding a mention in this subsection.

Results

My biggest criticism concerns Figures 3, 4, 5, and 6. The font is inappropriate, and the units on the Y-axis are not in accordance with the journal's requirements. I suggest presenting homogeneous groups above each bar on the graphs, as determined in the statistics. In the legends of the figures, please add the number of N values upon which the presented data were based.

 

Discussion

The discussion is quite interesting, although, similar to the "Introduction," it relies on rather dated studies. It would be beneficial to enrich it with the latest scientific findings.

 

Conclusions

The conclusions are properly drafted.

 

 

Comments on the Quality of English Language

General sentence structure needs to be addressed throughout.

 

 

Comments on the Quality of English Language

General sentence structure needs to be addressed throughout. 

Author Response

Reviewer 1

SPECIFIC COMMENTS

 

Abstract

The abstract is properly edited.

 

Introduction

The introduction is interesting and provides essential information regarding the significance of cyanobacteria. It may be worthwhile to delve further into the utilization of cyanobacteria in biofuel production and as fertilizers. This would underscore the importance of the conducted research, demonstrating the significance of cyanobacteria in the economy. I understand that a sentence on this topic is already included in the introduction, but it might be beneficial to add the latest statistical data on the use of cyanobacteria in tons and the quantity of biofuels produced from them. It would also be valuable to mention the leading countries in this field worldwide. I believe this is important, especially considering that the authors cite limited and outdated literature. Only 16 references pertain to studies from the last 10 years, including 6 from 2020 and above. This is a weakness of the manuscript.

Response: the authors appreciate very much the remarks of the reviewer and the suggestions concerning the literature review. Some literature was included in introduction including latest papers focusing the biotechnological applications of cyanobacteria in the field of biofuel.

 

page 2, line 86

The introduction is quite engaging; however, the final paragraph lacks a clearly stated research objective, and no research hypothesis is posited.

Response: we thank the reviewer for the remark. The last paragraph was modified in order to state the objectives and hypothesis of the study.

 

Materials and Methods

Chapter 2. Materials and Methods is well described. In subsection 2.5. Statistical Analysis, if the authors incorporate the suggestion for improving the graphs, I propose marking homogeneous groups for the mean bars and adding a mention in this subsection.

Response: the authors made the changes according to reviewer suggestion.

 

Results

My biggest criticism concerns Figures 3, 4, 5, and 6. The font is inappropriate, and the units on the Y-axis are not in accordance with the journal's requirements. I suggest presenting homogeneous groups above each bar on the graphs, as determined in the statistics. In the legends of the figures, please add the number of N values upon which the presented data were based.

Response: the graphs were modified following the suggestions of the reviewer, font style to “arial” and size 8. Furthermore, the scale of the y-axis was modified to be more uniform in all graphs. Furthermore, homogeneous groups above each bar on the graphs and by color.

 

Discussion

The discussion is quite interesting, although, similar to the "Introduction," it relies on rather dated studies. It would be beneficial to enrich it with the latest scientific findings.

Response: we thank the reviewer for the remark. Unfortunately, much of the research related with stability of microcystins with physico-chemical drivers was done in the past. We did an extensive search on the topic and no recent studies were found. Recent studies tackle mostly biodegradation and common water treatment chemicals (chlorination) and oxidation reactions using variety of catalysts (metal and carbon nanomaterials) which conditions are poorly related with the conditions tested in this work. Nevertheless, a few updated studies were found which were included in the manuscript

 

Conclusions

The conclusions are properly drafted.

 

Comments on the Quality of English Language

General sentence structure needs to be addressed throughout.

Response: the authors did a throughout revision of English sentence structure and improved the quality of the text.

Reviewer 2 Report

Comments and Suggestions for Authors

This study investigates the characteristics of cyanotoxin degradation by two types of cyanobacteria in response to external environmental factors (heat, UV light, ozone and sunlight). This is a topic of interest during times when concerns arise about the ecological harm and potential health risks posed by cyanotoxins. Overall, the experimental results using lyophilized biomass (figs 3 and 5) require validation of reproducibility or reinterpretation of the findings.

 - In the abstract, it's better not to capitalize "Cyanobacteria Harmful Algal Blooms."

- Keep the abstract concise and focused on results. For example, the following can be deleted: "Despite the concerns raised by these microorganisms in water bodies, cyanobacterial biomasses are also considered interesting sources of compounds with biotechnological application, being excellent soil amendments and plant biostimulants." Methodology details can be omitted.

- "M. aeruginosa" should be in italics (Line 121).

- Provide LC-MS manufacturer information.

- It's recommended to delete Table 2, which details the specific operating conditions of the mass spec, from the main text. It can be provided as supplementary material.

- Please ensure consistency in the order of listing the treatments for cyanotoxin degradation: heat, UV, ozone, sunlight.

- Is there a specific reason for applying a wavelength of 253.7 nm for UV light? An explanation is needed, as this wavelength is not typically for organic material degradation.

- LC-MS chromatograms presented in Fig. 1 & 2 should be provided as supplementary material (not in main text).

- In Fig. 3, the concentration of MC-LR after 2 hours of UV treatment appears to be twice campared to that of control, Given such low reproducibility, statistically significant decreases of 11% and 32% due to sunlight exposure need to be explained. Additionally, the significant degradation effect observed with 12 hours of UV light exposure should be addressed. Particularly, there needs to be a valid explanation for the low reproducibility of the UV experiments.

- Correct the misspelling of the figure number (Line 209).

- You mentioned “No significant changes were verified in this concentration when the freeze dried material was exposed to the different treatments except to thermal heating (50 ºC) for 12 h and UV light (2 h). These two treatments led to a decrease in the toxin amount to respectively 66% and 62% of the control (Figure 5; supplementary material 2, Table S3).” However concentration (25.6 ug/g) after UV 5h was lower that that (25.8 ug/g) of UV 2h (fig 5). And deviation of experiments in sunlight 2h exposure is too big to verify experimental reproducibility.

- Avoid lengthy repetition of experimental results in the discussion (lines 232-247). Furthermore, verbose explanations of indirectly related previous research findings should be deleted for the interpretation of the research results, and only directly relevant prior studies should be presented for explanation (lines 256-290).

Comments on the Quality of English Language

The English is at a level understandable to readers. However, some expressions may need to be addressed for clarity, and it would require a thorough review by the author themselves.

Author Response

Reviewer 2

 

Comments and Suggestions for Authors

This study investigates the characteristics of cyanotoxin degradation by two types of cyanobacteria in response to external environmental factors (heat, UV light, ozone and sunlight). This is a topic of interest during times when concerns arise about the ecological harm and potential health risks posed by cyanotoxins. Overall, the experimental results using lyophilized biomass (figs 3 and 5) require validation of reproducibility or reinterpretation of the findings.

Response: the authors thank the reviewer the remarks. Variability in the data was related mostly due to the extraction process of toxins from the cyanobacterial biomass. It involves several steps and small shortcomings in some of the steps could contribute to some variation in the data.

 

 - In the abstract, it's better not to capitalize "Cyanobacteria Harmful Algal Blooms."

Response: Thank you for the remark, changes were made.

 

- Keep the abstract concise and focused on results. For example, the following can be deleted: "Despite the concerns raised by these microorganisms in water bodies, cyanobacterial biomasses are also considered interesting sources of compounds with biotechnological application, being excellent soil amendments and plant biostimulants." Methodology details can be omitted.

Response: The introductory sentence was deleted but we kept the methodology information as we consider valuable for the reviewer to have a glimpse on the approach employed. This is not interfering with the length of the abstract and its content.

 

- "M. aeruginosa" should be in italics (Line 121).

Response: corrected

 

- Provide LC-MS manufacturer information.

Response: information added

 

- It's recommended to delete Table 2, which details the specific operating conditions of the mass spec, from the main text. It can be provided as supplementary material.

Response: information moved to supplementary material

 

- Please ensure consistency in the order of listing the treatments for cyanotoxin degradation: heat, UV, ozone, sunlight.

Response: we thank the remark of the reviewer, a throughout revision of the manuscript was performed.

 

- Is there a specific reason for applying a wavelength of 253.7 nm for UV light? An explanation is needed, as this wavelength is not typically for organic material degradation.

Response: this was the wavelength available in the lab. Furthermore, high energy, lower wavelength (254 nm) showed to be effective in degrading microcystins as described by Thirumavalavan et al (2012). This paper was added to the manuscript and UV wavelength discussed.

 

- LC-MS chromatograms presented in Fig. 1 & 2 should be provided as supplementary material (not in main text).

Response: we believe the chromatograms are necessary in the main text, since describe how the data was obtained and the reliability of the analysis.

 

- In Fig. 3, the concentration of MC-LR after 2 hours of UV treatment appears to be twice compared to that of control, Given such low reproducibility, statistically significant decreases of 11% and 32% due to sunlight exposure need to be explained. Additionally, the significant degradation effect observed with 12 hours of UV light exposure should be addressed. Particularly, there needs to be a valid explanation for the low reproducibility of the UV experiments.

Response: Indeed, we notice increased variations in a few replicate samples, which we attribute to the process of extraction of cyanotoxins from biomass. The process eventually need to be optimized to minimize variations. However, only few samples seemed to be affected by this variability and majority of data seem to be very consistent and reproducible, being SD less than 15%. Whenever homogeneity of variances was not met, non-parametric analysis was employed (Brown-Forsythe Welch’s ANOVA followed by unpaired t-test). The concentration of MC-LR after 2 hours of UV treatment differs from all other results because of the great variability. In order to minimize the effect of variation a non-parametric test was used.

 

- Correct the misspelling of the figure number (Line 209).

Response: corrected

 

- You mentioned “No significant changes were verified in this concentration when the freeze dried material was exposed to the different treatments except to thermal heating (50 ºC) for 12 h and UV light (2 h). These two treatments led to a decrease in the toxin amount to respectively 66% and 62% of the control (Figure 5; supplementary material 2, Table S3).” However, concentration (25.6 ug/g) after UV 5h was lower that (25.8 ug/g) of UV 2h (fig 5). And deviation of experiments in sunlight 2h exposure is too big to verify experimental reproducibility.

Response: except in one situation (CYN sunlight 2h exposure), in which variability is affecting interpretation of that specific condition, we do not consider there is a problem of experimental reproducibility. Indeed, variance (SD) determine the final statistical results and significant variations were detected in an experimental group with a mean value which is higher than another experimental group. In both situations SD was less than 3% of the respective means (UV 2h - 25.8 ± 0.3 ug/g and UV 5h - 25.5 ± 0.8 ug/g), so we do not consider there is a problem of experimental reproducibility.

 

- Avoid lengthy repetition of experimental results in the discussion (lines 232-247). Furthermore, verbose explanations of indirectly related previous research findings should be deleted for the interpretation of the research results, and only directly relevant prior studies should be presented for explanation (lines 256-290).

Response: The discussion was revised and experimental results in this section was reduced. recent literature related to the subject was included, as suggested by reviewer 1.

 

Comments on the Quality of English Language

The English is at a level understandable to readers. However, some expressions may need to be addressed for clarity, and it would require a thorough review by the author themselves.

Response: a thorough review was carried out as suggested.

 

Reviewer 3 Report

Comments and Suggestions for Authors

Dear Authors,

the presented study is very interesting, and in my opinion deserves being published.

I read it with interest and had very few suggestions how to improve the manuscript (please see below):

The Introduction is very comprehensive and informative, and generally very well written. It reads like an interesting piece of text.

Materials and Methods are also well described. All sub-chapters provide the necessary details and all information that would be useful for someone that would like to repeat the experiment.

l. 152-153: this fragment should probably be a part of a longer sentence

Results are summarized in the form of six graphs and in my opinion all of them are needed. The statistical significance of the results is shown by asterisks by the relevant bars in the graphs.

Discussion (its initial part) definitely needs refining. The first three paragraphs repeat the description of the results, which is pointless. Either do not describe the results again and only briefly mention them, or consider combining the Results and Discussion section.

The sub-chapter “Microcystin-LR decomposition” begins with some information regarding the stability of this toxin in the environment. If you could find similar information regarding the other toxin, you could use this information to begin the Discussion more generally than starting with results’ description.

l. 321: H2O2 – “2” should be lowercase

Conclusions

l. 354-356: this sentence is grammatically incorrect

Comments on the Quality of English Language

The language is genererally good. There are some very few issues, but nothing that would make it difficult to understand the text and would not be easily correctedd during the production process.

Author Response

Reviewer 3

 

Comments and Suggestions for Authors

Dear Authors,

the presented study is very interesting, and in my opinion deserves being published.

I read it with interest and had very few suggestions how to improve the manuscript (please see below):

 

The Introduction is very comprehensive and informative, and generally very well written. It reads like an interesting piece of text.

Materials and Methods are also well described. All sub-chapters provide the necessary details and all information that would be useful for someone that would like to repeat the experiment.

 

1. 152-153: this fragment should probably be a part of a longer sentence

Response: we thank the reviewer for the remark. Sentence corrected.

 

Results are summarized in the form of six graphs and in my opinion all of them are needed. The statistical significance of the results is shown by asterisks by the relevant bars in the graphs.

 

Discussion (its initial part) definitely needs refining. The first three paragraphs repeat the description of the results, which is pointless. Either do not describe the results again and only briefly mention them, or consider combining the Results and Discussion section.

Response: we thank the remarks of the reviewer. The discussion was reviewed according to the reviewers suggestions. First part was reduced, results description was kept short and discussion includes now a few more literature related to the topic.

 

The sub-chapter “Microcystin-LR decomposition” begins with some information regarding the stability of this toxin in the environment. If you could find similar information regarding the other toxin, you could use this information to begin the Discussion more generally than starting with results’ description.

Response: Thank you, we tried to keep the paragraph of results short. Yet we consider that this information needs to be in the discussion to facilitate the reading and comprehension of the discussion.

 

1. 321: H2O2 – “2” should be lowercase

Response: yes thank you

 

Conclusions

1. 354-356: this sentence is grammatically incorrect

Response: thank you for the remark. Sentence corrected.

 

Comments on the Quality of English Language

The language is genererally good. There are some very few issues, but nothing that would make it difficult to understand the text and would not be easily correctedd during the production process.

Round 2

Reviewer 2 Report

Comments and Suggestions for Authors

In this work, some of the data show low reliability due to the limited sample size, but overall, it is possible to compare the toxins degradation capabilities that the author aims to claim.