Response of Antioxidant Enzyme Activities of the Green Microalga Chlorococcum sp. AZHB to Cu²⁺ and Cd²⁺ Stress

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

 

The study describes the “Response of oxidase activities of the green microalga Chlorococcum sp. AZHB to Cu2+ and Cd2+ stress”. The research topic that the authors have researched is interesting. The authors show good results and the manuscript is prepared at a satisfactory level in general, however, some minor revisions are needed to be addressed before publication.

 

1) The authors reported that by increasing the concentration of Cu2+ and Cd2+ from 0 to 200 mg/L the MDA content also increases, While increasing the concentration of the Cu2+ and Cd2+ from 0.1 mg/L and 5 mg/L, respectively, decreases the activity of catalase (CAT). In the abstract section, the authors suggest the defense mechanisms of Chlorococcum sp. AZHB to heavy metals should be involved in the improvement of antioxidant enzyme activity in algal cells.

The concluded remarks are contradictory due to the following reasons;

a) The authors reported in the result and discussion section that the higher the MDA content higher will be the oxidative damage to the membrane system of the cell and even it causes death.

b) CAT transforms O2 and H2O2 into less active substances and reduces or eliminates the attack on membrane lipids which thus could be protected without peroxidation. However, at the concentration of the Cu2+ and Cd2+ from 0.1 mg/L and 5 mg/L, respectively, CAT activity decreases which mean no transformation into less active materials occurs.

By taking into inconsideration the above two statements and reasons how the authors concluded that the defense mechanism of Chlorococcum sp. AZHB to heavy metals is due to the improvement of antioxidant enzyme activity in algal cells.

 

2) Table 1. Is not clear. Overlap of number with the data is there. 

 

3) Conclusion part is not written clearly and need rewriting. 

Author Response

The study describes the “Response of oxidase activities of the green microalga Chlorococcum sp. AZHB to Cu²⁺ and Cd²⁺ stress”. The research topic that the authors have researched is interesting. The authors show good results and the manuscript is prepared at a satisfactory level in general, however, some minor revisions are needed to be addressed before publication.

Authors’ response: We sincerely thank the Reviewer #1 for his/her positive comments, and for his/her time and effort in reviewing our manuscript and providing the constructive and useful comments.

 

1) The authors reported that by increasing the concentration of Cu²⁺ and Cd²⁺ from 0 to 200 mg/L the MDA content also increases, While increasing the concentration of the Cu²⁺ and Cd²⁺ from 0.1 mg/L and 5 mg/L, respectively, decreases the activity of catalase (CAT). In the abstract section, the authors suggest the defense mechanisms of Chlorococcum sp. AZHB to heavy metals should be involved in the improvement of antioxidant enzyme activity in algal cells.

The concluded remarks are contradictory due to the following reasons;

1. a) The authors reported in the result and discussion section that the higher the MDA content higher will be the oxidative damage to the membrane system of the cell and even it causes death.
2. b) CAT transforms O₂ and H₂O₂ into less active substances and reduces or eliminates the attack on membrane lipids which thus could be protected without peroxidation. However, at the concentration of the Cu²⁺ and Cd²⁺ from 0.1 mg/L and 5 mg/L, respectively, CAT activity decreases which mean no transformation into less active materials occurs.

By taking into inconsideration the above two statements and reasons how the authors concluded that the defense mechanism of Chlorococcum sp. AZHB to heavy metals is due to the improvement of antioxidant enzyme activity in algal cells.

Authors’ response: Thanks for the comment. Yes, enzyme activity of CAT decreased when the concentrations of Cu²⁺ and Cd²⁺ were greater than 0.1 and 5 mg/L, respectively. Even so, the enzyme activity of CAT in those treatments were significantly higher than that in control without the exposure of heavy metals (Figure 4). Thus, we concluded that the defense mechanism of Chlorococcum sp. AZHB to heavy metals is due to the improvement of antioxidant enzyme activity in algal cells.

 

Obviously, the response of enzyme activity of CAT was concentration dependent, and the response of CAT was not consistent with other enzymes like POD and SOD. The response of activity of CAT showed an inverse-V shape with the increase of metal concentrations, which were also found in previous studies (e.g., ref [27]). The possible reason might be attributable to the inhibition of CAT enzyme synthesis or the change in assembly of enzyme subunits at extremely high concentration of heavy metals. We have mentioned these in the third paragraph in the Results and Discussion section.

 

2) Table 1. Is not clear. Overlap of number with the data is there.

Authors’ response: We have modified the table and its footnote.

 

3) Conclusion part is not written clearly and need rewriting.

Authors’ response: Thanks for the comment. Revised as suggested.

Author Response File: Author Response.docx

Reviewer 2 Report

The concept of the study is clear and the draft has written according to the journal's format. Introduction is brief enough for understand the aim. Methodology section is clear, however few fuzzy statements are quoted in this article and seems. The specific comments are appended here for correction before publication. I recommend the manuscript can be accepted after minor revision.

 

1. How the authors identify and confirm the algal strain as Chlorococcum sp.?

2. The authors should be clearly noticed the quantity (How much?) mL of stock solution which was taken for the experiment?

3. How to measure the different concentration of metal stock solution?

4. How to determine the enzyme activity? No detailed methodology in the manuscript.

 

5. How to utilize the Cu and Cd metals by the algal cell? Any specific mechanisms behind it. The author should give proper schematic explanation.

 

Author Response

The concept of the study is clear and the draft has written according to the journal's format. Introduction is brief enough for understand the aim. Methodology section is clear, however few fuzzy statements are quoted in this article and seems. The specific comments are appended here for correction before publication. I recommend the manuscript can be accepted after minor revision.

Authors’ response: We sincerely thank the Reviewer #2 for the time and effort in reviewing our manuscript and providing many useful and constructive comments.

 

1. How the authors identify and confirm the algal strain as Chlorococcum sp.?

Authors’ response: The green microalgal strain Chlorococcum sp. AZHB was collected and isolated from a sewage treatment plant of Arizona, USA. The stain was then identified to the genus level according to its morphology, and maintained in BG11 medium in the laboratory.

 

2. The authors should be clearly noticed the quantity (How much?) mL of stock solution which was taken for the experiment?

Authors’ response: The stock solutions of copper (i.e., 200 and 20 mg/L) and stock solutions of cadmium (i.e., 2000 and 50 mg/L) were prepared respectively. These stocks were then diluted to their respective working concentrations in 150 mL test solutions with BG11 medium.

 

3. How to measure the different concentration of metal stock solution?

Authors’ response: The powders of heavy metals were weighed with a balance, and then dissolved the metals to the BG11 medium to obtain the stocks.

 

4. How to determine the enzyme activity? No detailed methodology in the manuscript.

Authors’ response: Thanks for the comment. Because the determination of enzyme activities has already detailed methods in previous studies, we only show those necessary information for understanding this work with the purpose to save the space of main text. Please see the detailed information in the related refs, i.e., refs [20] and [21].

 

5. How to utilize the Cu and Cd metals by the algal cell? Any specific mechanisms behind it. The author should give proper schematic explanation.

Authors’ response: We appreciate the suggestion from the reviewer, and have added a new figure (i.e., Figure 5) to show the response mechanisms of the algal species to the exposure of heavy metals.

Author Response File: Author Response.docx

Reviewer 3 Report

In this article, the author studied the physiological changes after the green microalgal Chlorococcum sp. AZHB came into contact with heavy metals. By observing the algae species in Cu²⁺ or Cd²⁺ to cultivate 10 days of oxidase activity in different concentrations, it shows that with the increase of the concentration of Cu²⁺ and Cd²⁺ peroxidase (POD) and superoxide dismutase (SOD) activity increased. The inhibitory effect of  Chlorococcum sp. AZHB may be related to improving the activity of antioxidant enzyme in algae cell. There are fewer studies on algae cells in heavy metal environments, so the study is more meaningful. However, before accepting, several small problems need to be solved. For details, see the following comments.

1. The journal name of the reference should be written obliquely.

2. You should follow the manuscript format of this journal. Including references, the format does not show any consistency.

Author Response

Reviewer 3:
In this article, the author studied the physiological changes after the green microalgal Chlorococcum sp. AZHB came into contact with heavy metals. By observing the algae species in Cu²⁺ or Cd²⁺ to cultivate 10 days of oxidase activity in different concentrations, it shows that with the increase of the concentration of Cu²⁺ and Cd²⁺ peroxidase (POD) and superoxide dismutase (SOD) activity increased. The inhibitory effect of Chlorococcum sp. AZHB may be related to improving the activity of antioxidant enzyme in algae cell. There are fewer studies on algae cells in heavy metal environments, so the study is more meaningful. However, before accepting, several small problems need to be solved. For details, see the following comments.

Authors’ response: We welcome and appreciate such positive comments from the Reviewer #3, and would like to thank the reviewer for his/her great effort in going through our manuscript and providing the supportive and useful comments.

 

1. The journal name of the reference should be written obliquely.

Authors’ response: Revised as suggested.

 

2. You should follow the manuscript format of this journal. Including references, the format does not show any consistency.

Authors’ response: Thanks for the suggestion. We have formatted the manuscript according to the requirement of the journal.

Author Response File: Author Response.docx