Photophysiological Characterization of Phytoplankton by Measuring Pigment Production Rates: A Description of Detail Method and a Case Study
Round 1
Reviewer 1 Report
Comments and Suggestions for Authors
The paper presented by Kang et al. gives an insight on the application of two HPLC methodologies to detect pigments in phytoplankton. The previous processes before the analysis used proved to have better results for the detection and quantification of phytoplankton pigments. Methodologies applied are well explained, English is well written. Discussion gives detailed information for other researches to apply the methodologies with good advices to improve results. This research helps other investigations for the importance of pigments in aquatic environments, providing high relevance of their role.
Minor revisions should be applied.
- Improve figure 4 resolution. Authors could put the two graphs vertically for better visualization. The same could be applied to figure 6.
- Check that in situ is in italic.
- For figure 9, X-axis limits could be from 0.2 or 0.3 to 1.5; this could make the points to be better distributed.
Author Response
Reviewer 1
The paper presented by Kang et al. gives an insight on the application of two HPLC methodologies to detect pigments in phytoplankton. The previous processes before the analysis used proved to have better results for the detection and quantification of phytoplankton pigments. Methodologies applied are well explained, English is well written. Discussion gives detailed information for other researches to apply the methodologies with good advices to improve results. This research helps other investigations for the importance of pigments in aquatic environments, providing high relevance of their role.
Minor revisions should be applied.
- Improve figure 4 resolution. Authors could put the two graphs vertically for better visualization. The same could be applied to figure 6.
--> Based on your comments, we have adjusted Figures 4 and 6 to be displayed vertically.
- Check that in situ is in italic.
--> All instances of the term "in-situ" in the text have been changed to italic form.
- For figure 9, X-axis limits could be from 0.2 or 0.3 to 1.5; this could make the points to be better distributed.
--> The range of the x-axis has been modified to span from 0.3 to 1.5 ng C L–1 h–1. In addition, the range of the y-axis was also modified for better distribution of points.
Author Response File: 
Author Response.pdf
Reviewer 2 Report
Comments and Suggestions for AuthorsThis manuscript is innovative in the measurement of pigment production rate by introducing isotopic labelling, but the manuscript has some problems and revisions are recommended.
1. how the authors remove the effect of 13C in the natural background on the measurement
2. The article uses HPLC for pigment quantification, but why not use mass spectrometry for quantification, please add a part in the article to explain that mass spectrometry is more suitable for quantification than HPLC. For example, the amount of 13C mixed in is uncertain.
3. how did the authors determine the composition of the different algae in the sample, using sequencing?
4. in line 271, the authors state that the previous study of 17% inorganic carbon was the maximum dose allowed. But how did the authors determine that 15% inorganic carbon was the appropriate concentration?
5. It is suggested to cite the literature to increase the number of pigment types produced by different species of algae to be analysed.
6. how the authors verified the efficiency of HPLC in separating the pigments during continuous injection.
Comments on the Quality of English LanguageSuggests requesting a professional structure for linguistic embellishment
Author Response
Reviewer 2
This manuscript is innovative in the measurement of pigment production rate by introducing isotopic labelling, but the manuscript has some problems and revisions are recommended.
- how the authors remove the effect of 13C in the natural background on the measurement
--> A factor that can affect the delta 13C value of natural background in the procedure of pigment production analysis is the solvent used in HPLC analysis. Thus, we conducted an experiment to determine the effect of solvent on the natural background (2.1 section; line 86-95), and the results are shown in Figure 4. STD in Figure 4 is the result of measuring the isotope value directly on the authentic standard of chlorophyll-a (Sigma-Aldrich) without any treatment such as HPLC analysis. The rest is the result of isotope analysis for the same authentic standard of chlorophyll-a (Sigma-Aldrich) separated by a fraction collector after HPLC analysis (based on the ZM and JM) at different injection volumes (100 μL – 500 μL). Based on these results, we determined that effect of 13C in the natural background on the measurement is negligible.
- The article uses HPLC for pigment quantification, but why not use mass spectrometry for quantification, please add a part in the article to explain that mass spectrometry is more suitable for quantification than HPLC. For example, the amount of 13C mixed in is uncertain.
--> If there is only a single pigment in the sample, quantification using mass spectrometry would be more accurate. However, since samples obtained from the field contain various pigments, separation of these pigments must be done first. So, we separated the pigments using the HPLC and then collected each pigment through a fraction collector. Afterwards, the delta 13C value of each separated pigment was analyzed by mass spectrometry (Finnigan Delta + XL mass spectrometer in “Stable Isotope Laboratory of the University of Alaska Fairbanks, USA”) (Section 2.3).
- how did the authors determine the composition of the different algae in the sample, using sequencing?
--> We determined the composition of the different algae in the sample using the ratio of marker pigments to chl-a reported in similar study region. The table (Lee et al., 2011) below shows the ratio of pigments we used.
|
Taxa |
Perid |
But-fuco |
Fuco |
Hex-fuco |
Neo |
Pras |
Viola |
Allo |
Lut |
Zea |
Chl-b |
Chl-a |
|
Prasino |
0 |
0 |
0 |
0 |
0.3768 |
0.1413 |
0.2165 |
0 |
0.0843 |
0 |
0.2807 |
1 |
|
Dino |
0.7527 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
1 |
|
Crypto |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0.1927 |
0 |
0 |
0 |
1 |
|
Prymne |
0 |
0 |
0 |
1.7139 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
1 |
|
Pelago |
0 |
0.5076 |
0.8354 |
0.2225 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
1 |
|
Chloro |
0 |
0 |
0 |
0 |
0.0756 |
0 |
0.0457 |
0 |
0.2253 |
0.0063 |
0.4255 |
1 |
|
Cyano |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0.1418 |
0 |
1 |
|
Bacilla |
0 |
0 |
0.5464 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
1 |
Finally, the phytoplankton community composition was derived using the CHEMTAX program based on this pigment ratio (Mackey et al., 1996).
References
*Lee, Y. W., Park, M. O., Kim, Y. S., Kim, S. S., & Kang, C. K. (2011). Application of photosynthetic pigment analysis using a HPLC and CHEMTAX program to studies of phytoplankton community composition. The Sea: JOURNAL OF THE KOREAN SOCIETY OF OCEANOGRAPHY, 16(3), 117-124.
* Mackey, M. D., Mackey, D. J., Higgins, H. W., & Wright, S. W. (1996). CHEMTAX-a program for estimating class abundances from chemical markers: application to HPLC measurements of phytoplankton. Marine Ecology Progress Series, 144, 265-283.
- in line 271, the authors state that the previous study of 17% inorganic carbon was the maximum dose allowed. But how did the authors determine that 15% inorganic carbon was the appropriate concentration?
--> In general, the injection amounts of carbon and nitrogen isotopes are recommended to be approximately 5-10% of the total inorganic carbon and nitrogenous nutrients in the ambient water to prevent overestimation (Dugdale and Goering 1967; Dugdale and Wilkerson 1986). However, when the experiment was first conducted at the corresponding injection concentration, the production rate could not be calculated because carbon isotopes were not sufficiently labeled in most minor pigments except chlorophyll-a. Subsequently, as the injection concentration of the carbon isotope reagent was raised to approximately 15-17%, it became possible to measure the productivity of the majority of significant pigments. However, according to your opinion, it was judged that it was problematic to say that only 15% concentration was the appropriate amount, so we revised the text to the 15-17% concentration range (line 300).
References
*Dugdale RC, Goering JJ (1967) Uptake of new and regenerated forms of nitrogen in primary productivity. Limnol Oceanogr 12:196–206
*Dugdale RC, Wilkerson FP (1986) The use of 15N to measure nitrogen uptake in eutrophic oceans; experimental considerations. Limnol Oceanogr 31:673–689
- It is suggested to cite the literature to increase the number of pigment types produced by different species of algae to be analysed.
--> We also agree with your opinion. Thus, related information is additionally suggested in the conclusion section (line 418-420; Table 5).
- how the authors verified the efficiency of HPLC in separating the pigments during continuous injection.
--> The efficiency of HPLC in separating pigments during continuous injection was verified by real-time comparing with the initial qualitative and quantitative analysis results of each sample. Although there was some variation in detection time, the separation efficiency was confirmed to be adequate for all samples (data were not shown).
Author Response File: Author Response.pdf
Reviewer 3 Report
Comments and Suggestions for AuthorsManuscript title:
Measuring pigment production rates of phytoplankton using HPLC pigment extraction method: A case study in the East/Japan Sea
Jae Joong Kang, Jun-Oh Min, Huitae Joo, Seok-Hyun Youn, and Sang Heon Lee
The manuscript is well-written and the subject is novel. Please answer the following comments:
1. The title needs to be redesigned to be more attractive to readers; it can begin like this ‘Characterization of phytoplankton …”
2. Line 31: In keywords, the HPLC is more appropriate, and (high-performance liquid chromatography) is not necessary.
3. The introduction should be improved by the addition of some paragraphs and good references regarding the importance of pigments, their biosynthesis, and their application in different fields.
4. Line 125: The subtitle “2.3. Extraction and Analysis for Phytoplankton Pigments for Production Assessment” is vague and needs to be rewritten grammatically.
5. A table summarizing a number of updated references can improve the paper.
Author Response
Reviewer 3
Measuring pigment production rates of phytoplankton using HPLC pigment extraction method: A case study in the East/Japan Sea
Jae Joong Kang, Jun-Oh Min, Huitae Joo, Seok-Hyun Youn, and Sang Heon Lee
The manuscript is well-written and the subject is novel. Please answer the following comments:
- The title needs to be redesigned to be more attractive to readers; it can begin like this ‘Characterization of phytoplankton …”
--> Based on your comments, the title has been changed to "Photophysiological characterization of phytoplankton by measuring pigment production rates: A description of detail method and a case study".
- Line 31: In keywords, the HPLC is more appropriate, and (high-performance liquid chromatography) is not necessary.
--> The content in parentheses following "HPLC" was removed in the keyword section.
- The introduction should be improved by the addition of some paragraphs and good references regarding the importance of pigments, their biosynthesis, and their application in different fields.
--> Based on your comments, we additionally described regarding the importance of pigments in the "introduction" (line 41-51).
- Line 125: The subtitle “2.3. Extraction and Analysis for Phytoplankton Pigments for Production Assessment” is vague and needs to be rewritten grammatically.
--> Subtitle 2.3 has been modified to "Extraction and Analysis of Phytoplankton Pigments for Production Assessment".
- A table summarizing a number of updated references can improve the paper.
--> We added a new table (Table 5) for summarizing updated references in our revised manuscript.
Author Response File: Author Response.pdf
Reviewer 4 Report
Comments and Suggestions for AuthorsThis well-written and organized manuscript presents the study of different analytic variables and their impact on the determination of phytoplankton pigment rates using liquid chromatography. Some minor modifications could be addressed in order to improve the quality and impact of this article, as suggested below.
It would be useful for readers and researchers interested in this field if the authors included more specific (numeric) results in the abstract. In my opinion, the results and conclusions presented in this section seem to be vague and do not highlight the study findings. I suggest restructuring the next paragraph by adding relevant results: “the results showed that the notable production rates of chl-b, an accessory pigment mainly attributed to prasinophytes, potentially due to restricted light availability. Prioritization of chl-b 25 production over primary production highlighted the potential impact of compensatory pigment-related activities on overall phytoplankton productivity. In conclusion, this study underscores the significance of directly quantifying pigment production rates to enhance our comprehension of phytoplankton photophysiology and the production mechanisms specific to various pigments”
Since the purpose of this study encompasses the evaluation and establishment of analytical methodologies to detect and quantify pigment production both ex-situ and in-situ, is strongly recommended to include a sub-section in materials and methods describing the statistical methodologies, including the number of samples analyzed, replicates, repetitions (if applicable), as well as the statistical programs, test, and parameters used. In the results section, the authors briefly describe this part, however, it should be better addressed in both methods and results.
Subsection 2.1 Carbon Stable Isotope Fractionation Experiment for Pigment Production states that chromatographic conditions are described in Table 1, however, these are detailed in Table 2.
The results presented in Table 3, and the findings stated in lines 184-185 “These findings 184 collectively indicate that variations in sample injection volume did not introduce over- or 185 underestimations in the analysis results” could be better supported by implementing a statistical analysis that correlated the volume injection, response, and significance variations.
Conclusions should be improved by adding precise findings rather than vague statements. It is necessary to include a critical analysis of the mentioned studies; limitations and future potential.
Comments on the Quality of English Language
Minor corrections are required
Author Response
Reviewer 4
This well-written and organized manuscript presents the study of different analytic variables and their impact on the determination of phytoplankton pigment rates using liquid chromatography. Some minor modifications could be addressed in order to improve the quality and impact of this article, as suggested below.
It would be useful for readers and researchers interested in this field if the authors included more specific (numeric) results in the abstract. In my opinion, the results and conclusions presented in this section seem to be vague and do not highlight the study findings. I suggest restructuring the next paragraph by adding relevant results: “the results showed that the notable production rates of chl-b, an accessory pigment mainly attributed to prasinophytes, potentially due to restricted light availability. Prioritization of chl-b production over primary production highlighted the potential impact of compensatory pigment-related activities on overall phytoplankton productivity. In conclusion, this study underscores the significance of directly quantifying pigment production rates to enhance our comprehension of phytoplankton photophysiology and the production mechanisms specific to various pigments”
--> Based on your comments, we have modified the suggested paragraph by adding relevant results (line 26-29).
Since the purpose of this study encompasses the evaluation and establishment of analytical methodologies to detect and quantify pigment production both ex-situ and in-situ, is strongly recommended to include a sub-section in materials and methods describing the statistical methodologies, including the number of samples analyzed, replicates, repetitions (if applicable), as well as the statistical programs, test, and parameters used. In the results section, the authors briefly describe this part, however, it should be better addressed in both methods and results.
--> Based on your recommendations, we have added statistical analysis (sub-section 2.5) to the Materials and Methods section (line 184).
Subsection 2.1 Carbon Stable Isotope Fractionation Experiment for Pigment Production states that chromatographic conditions are described in Table 1, however, these are detailed in Table 2.
--> The position and number of the table have been modified to fit the content.
The results presented in Table 3, and the findings stated in lines 184-185 “These findings collectively indicate that variations in sample injection volume did not introduce over- or underestimations in the analysis results” could be better supported by implementing a statistical analysis that correlated the volume injection, response, and significance variations.
--> We also agree with you. Thus, the area (i.e. “response”) according to each injection amount using the HPLC was obtained through three repeated experiments, and R.S.D. (i.e. “significance variations) was calculated to determine the variability and precision. If there is a better statistical analysis method that can support the results, please recommend it.
Conclusions should be improved by adding precise findings rather than vague statements. It is necessary to include a critical analysis of the mentioned studies; limitations and future potential.
--> We revised our conclusion with precise findings. Additionally, critical analysis by presenting the limitations of this study was added in the conclusion section (397-433).
Author Response File: Author Response.pdf
Round 2
Reviewer 2 Report
Comments and Suggestions for AuthorsThe author has answered my question very well and recommends receiving this manuscript.
