Colaconema formosanum, Sarcodia suae, and Nostoc commune as Fermentation Substrates for Bioactive Substance Production

Round 1

Reviewer 1 Report

The article studied by Meng-Chou Lee et al. investigated the fermentation of three algae species using Pseudoalteromonas haloplanktis, which could provide some useful information in the biofermentation process. However, I have several concerns related to its publication.

1- The abstract has to be re-written. It is too general without any data or numbers of the main results. 

2- Line 17, 'efficient, low cost, and green extraction and purification methodology', be careful with these statements. Did you compare the efficiency and cost to other generally used techniques? Do you have any data in your manuscript related to cost?

3- Line 21, 'a simple and widely available biorefinery process', what is the evidence to support this conclusion? Why it is simple? What are the currently used techniques for phycobiliproteins and mannose extraction? 

4- The introduction should be improved. The current status of using Pseudoalteromonas haloplanktis or other bacteria for high-value product production by fermentation should be introduced.

5-Line 77, 'until an OD600 of ~1.0 was reached, why OD600~1 was selected?

6- Line 155-156, standard deviations for numbers should be provided when there are no tables or figures to show the original data. 

7-The growth of Pseudoalteromonas haloplanktis should be provided for the fermentation process.

8-Figure 1&2, what is the meaning to test the points after 24 h? already stable for three-time points. Why not test 2, 3, 4 h? Are you sure 6 h is the best fermentation time? not less than 6 h?

9 Line 216 'C. formosanum was used for further analysis.' why this alga was selected? Why not another two species? Why not investigate three of them?

10-Figure 6-10, Do not give notes for Label “physical…”: supernatant collected after strong 261 fragmentation by FAST–PREP 24. Change the label in figures such as 'supernatant'. DO NOT use “physical…” as the label.

11-Figure 6-10, the figure X-axises are quite confusing. Explain the time from 0-6-0-6.

12-What do you want to tell people by showing Fig. 11 with only one sentence in the text?

13-The discussion should be improved carefully. It is too short without deep comparisons with available references. 

Author Response

Reviewer’s comment (1).

The abstract has to be re-written. It is too general without any data or numbers of the main results. 

Response: Thank you for raising this important point. We have rewritten our manuscript accordingly. (Line 22-28).

Reviewer’s comment (2).

Line 17, 'efficient, low cost, and green extraction and purification methodology', be careful with these statements. Did you compare the efficiency and cost to other generally used techniques? Do you have any data in your manuscript related to cost?

Response: Thanks for your valuable suggestion. We have revised our manuscript

as ‘Therefore, an un-complicated green extraction and purification methodology capable of recovering bio-compounds from algal biomass is of extreme importance in commercial production.’ (Line 16-18).

Reviewer’s comment (3).

Line 21, 'a simple and widely available biorefinery process', what is the evidence to support this conclusion? Why it is simple?

Response: Thanks for your valuable suggestion. We have revised our manuscript

as ‘In this study, we describe a feasible biorefinery process for the production of phycobiliproteins and mannose from marine macroalgae and cyanobacteria.’ (Line 26-27).

 

Reviewer’s comment (4).

Line 21, What are the currently used techniques for phycobiliproteins and mannose extraction?

Response: Thanks for your valuable suggestion. We have added several sentences in introduction section. The changes we have made are marked in yellow in the following (Lines 43-46).

In tradition, physical high pressure and chemical solvent extract method are often used to extract biologically active compounds, for instance solvent systems, sonication, ultrasound, microwave, enzymatic, super-critical fluid, and subcritical water.

Reviewer’s comment (5).

The introduction should be improved. The current status of using Pseudoalteromonas haloplanktis or other bacteria for high-value product production by fermentation should be introduced.

Response: Thanks for your valuable suggestion. We have rewritten introduction section accordingly. The changes we have made are marked in yellow in the following (Lines 33-78).

Reviewer’s comment (6).

Line 77, 'until an OD600 of ~1.0 was reached, why OD600~1 was selected?

Response: Thanks for your valuable suggestion. We have revised our manuscript as ‘For fermentation, the cultivated condition was converted into at 22°C for 12 hours (log phase) (Appendix A), and harvested with 5000 x g centrifugation for 10 min. The cultural broth was washed out twice with distilled water before application.’ (Line 97-100).

Reviewer’s comment (7).

Line 155-156, standard deviations for numbers should be provided when there are no tables or figures to show the original data.

Response: Done. We have revised our manuscript accordingly.

Reviewer’s comment (8).

The growth of Pseudoalteromonas haloplanktis should be provided for the fermentation process.

Response: We thank reviewer’s question and understand the reviewer’s point. Although may not provide the optimal growth of Pseudoalteromonas haloplanktis, we clearly demonstrated the feasibility of using bacteria to ferment the algae and therefore provide better extract conditions.

Reviewer’s comment (9).

Figure 1&2, what is the meaning to test the points after 24 h? already stable for three-time points. Why not test 2, 3, 4 h? Are you sure 6 h is the best fermentation time? not less than 6 h?

Response: We thank reviewer’s question and understand the reviewer’s point. Although the precise optimal fermentation time is unclear in this study, we clearly demonstrated the feasibility of using bacteria to ferment the algae and therefore provide better extract conditions.

Reviewer’s comment (10).

Line 216 'C. formosanum was used for further analysis.' why this alga was selected? Why not another two species? Why not investigate three of them?

Response: Thanks for your valuable suggestion. We have added several sentences in our manuscript accordingly. (Lines 239-242).

From the previous fermentation experiment, C. formosanum showed significantly higher production of PE, PC, APC, Chl a and reducing sugar at 6 h compared to S. suae and N. commune. To further promote the production of those high-value bioactive substances from algae, C. formosanum was used for further analysis.

Reviewer’s comment (11).

Figure 6-10, Do not give notes for Label “physical…”: supernatant collected after strong fragmentation by FAST–PREP 24. Change the label in figures such as 'supernatant'. DO NOT use “physical…” as the label.

Response: Done. We have revised our manuscript accordingly.

Reviewer’s comment (12).

Figure 6-10, the figure X-axises are quite confusing. Explain the time from 0-6-0-6.

Response: Done. We have revised our manuscript accordingly.

Reviewer’s comment (13).

What do you want to tell people by showing Fig. 11 with only one sentence in the text?

Response: Thanks for your valuable suggestion. We have added several sentences in Fig. 11 accordingly. (Lines 322-325).

Figure 11. The mannose variation of Colaconema formosanum, Sarcodia suae and Nostoc commune assayed by HPLC analysis. The algae were assayed by HPLC before (a) and after (b) fermentation. Fermented C. formosanum, S. suiae and N. commune were diluted 100 times before passing the HPLC column.

Reviewer’s comment (14).

The discussion should be improved carefully. It is too short without deep comparisons with available references.

Response: Thanks for your valuable suggestion. We have rewritten discussion section accordingly. The changes we have made are marked in yellow in the following (Lines 333-393).

Thank you for considering once again this manuscript.

Author Response File: Author Response.docx

Reviewer 2 Report

The manuscript provides a simple biorefinery process for the production of phycobiliproteins which is worth publishing. However, some questions may be needed to clarify before publishing. First of all, the phycobiliproteins were suggested in introductions to be pharmaceutical applications as even immunoassays which needed high purity. However, the products from the biorefinery process suggested in the manuscript were a mixture of phycobiliproteins and others polysaccharides, pigments, etc. Are there any suggestinet of downstream process after the suggested biorefinery process?

In the experiments, the fresh seaweed was collected and removed visible surface contaminants, was there any further sterilised process on the fresh seaweed? If not, may the remained microorganism attached on the fresh seaweed will have interacted with P.haloplanktis during fermentation and further affect the results? In figure 3, there was an obvious decrease of allophycocyanin concentrations during the fermentation of C.formosanum at 30 hours and also chlorophyll a at 36 hours. What are the reasons behind ? 

In discussion, it suggest a previous study of the extraction of PE from Gelidium pusillum, are there a comparison between the suggested process, e.g. which one is better? 

Author Response

We appreciate the reviewers’ comments on our manuscript and believe the input helped us improve the quality of this paper. We have made all the suggestions and corrections according to reviewers’ suggestions. Considering the reviewers’ comments, please see below for our responses to the reviewers’ comments.

Reviewer’s comment (1).

First of all, the phycobiliproteins were suggested in introductions to be pharmaceutical applications as even immunoassays which needed high purity. However, the products from the biorefinery process suggested in the manuscript were a mixture of phycobiliproteins and others polysaccharides, pigments, etc. Are there any suggestinet of downstream process after the suggested biorefinery process?

Response: Thanks for your valuable suggestion. We have rewritten discussion section accordingly. The changes we have made are marked in yellow in the following (Lines 385-388).

Notably, due to the possibility of the extract products to be used in pharmaceutical or research fields, purification and sterile processes such as salting-out and filtration are necessary after the biorefinery process, which is our further research point.

Reviewer’s comment (2).

In the experiments, the fresh seaweed was collected and removed visible surface contaminants, was there any further sterilised process on the fresh seaweed? If not, may the remained microorganism attached on the fresh seaweed will have interacted with P.haloplanktis during fermentation and further affect the results?

Thanks for your valuable suggestion. We have added several sentences in materials and methods section. The changes we have made are marked in yellow in the following (Lines 93-97).

Before experiments, algae samplings were cultured in vessels with sterile media and a clean growth chamber. During cultivation, the pollution by other algae or bacteria was absent. Since the sterilized process could destroy the biochemical compounds, the algae were fermenting in a fresh situation, and we hypothesize that the main fermented reaction from the advantage bacteria P. haloplanktis.

Reviewer’s comment (3).

In figure 3, there was an obvious decrease of allophycocyanin concentrations during the fermentation of C.formosanum at 30 hours and also chlorophyll a at 36 hours. What are the reasons behind?

Response: We thank the reviewer’s question, inhere we regarded that the decrease of pigment was due to the sampling collected under a heterogeneity situation.

Reviewer’s comment (4).

In discussion, it suggest a previous study of the extraction of PE from Gelidium pusillum, are there a comparison between the suggested process, e.g. which one is better?

Response: Thanks for your valuable suggestion. We have revised several sentences in discussion section accordingly. The changes we have made are marked in yellow in the following (Lines 335-359).

A previous study focused on the extraction of PE from Gelidium pusillum by enzymatic hydrolysis demonstrated that the mixture of enzymes (agarase, xylanase, and cellulase) at 25 °C for 5 h significantly improved the extraction yield of PE rather, compared to individual enzymes which resulted in low extraction efficiency (0.29 mg PE in each gram of dry algae) [14].

 

Thank you for considering once again this manuscript.

Author Response File: Author Response.docx

Reviewer 3 Report

In this manuscript, the authors present a process where three types of algae (Colaconema formosanum, Sarcodia suae, and Nostoc commune) are subjected to fermentation steps using Pseudoalteromonas haloplanktis ATCC 14393. This strain degraded the polysaccharides from the algae cell walls (cellulose, xylan or mannan fibrils, carrageenan and agar) and the combined fragmenting and fermenting method increased the extraction efficiency of phycobiliproteins and chlorophyll a. The bacteria further metabolized glucose and galactose to produce mannose. Although the fermentation needs to be optimized, the successful preliminary results obtained in this experimental study can pave the way for further experiments.

The bibliographic references are appropriate and up-to date. For consistency, use either the abbreviated or complete journal name (ex: Line 433 “Mol Biotechnol” and Line 435 “Molecular Biotechnology”). Review and correct the entire list.

 

 Please address the following comments/remarks:

 

1-     The authors should further detail the advantages of using P. haloplanktis instead of an enzymatic cocktail. 

2-     The authors should include a comment on the feasibility of the scale up of this process.

3-     Page 13 Line 319 – correct “xylanase” to “xylan”

4-     Page 14 – Remove:

“h: hour 

w: weight

g: gram 

mg: milligram 

L: liter”

from the list of abbreviations; the units have international symbols and are not “abbreviations”

5-     Page 14 - Line 385 Correct “Acknowledgments” to “Acknowledgements” 

 

 

 

Author Response

We appreciate the reviewers’ comments on our manuscript and believe the input helped us improve the quality of this paper. We have made all the suggestions and corrections according to reviewers’ suggestions. Considering the reviewers’ comments, please see below for our responses to the reviewers’ comments.

Reviewer’s comment (1).

The bibliographic references are appropriate and up-to date. For consistency, use either the abbreviated or complete journal name (ex: Line 433 “Mol Biotechnol” and Line 435 “Molecular Biotechnology”). Review and correct the entire list.

Response: Done. We have revised our manuscript accordingly.

Reviewer’s comment (2).

The authors should further detail the advantages of using P. haloplanktis instead of an enzymatic cocktail.

Response: Thanks for your valuable suggestion. We have revised several sentences in discussion section accordingly. The changes we have made are marked in yellow in the following (Lines 388-390).

In view of the success of fermentation in this research, we suggest that P. haloplanktis ATCC 14393 was an ideal species to ferment in algae, by contrast, the threshold in the culture of this bacteria was lower than enzyme collection.

Reviewer’s comment (3).

The authors should include a comment on the feasibility of the scale up of this process.

Response: Thanks for your valuable suggestion. We have revised several sentences in discussion section accordingly. The changes we have made are marked in yellow in the following (Lines 391-392).

Thus, P. haloplanktis ATCC 14393 has high potential to use in the mass-production industry of the algae, it is worthy to establish the scale-up technique of this process in the future.

Reviewer’s comment (4).

Page 13 Line 319 – correct “xylanase” to “xylan”

Response: Done. We have revised our manuscript accordingly.

Reviewer’s comment (5).

Page 14 – Remove:

“h: hour 

w: weight

g: gram 

mg: milligram 

L: liter”

from the list of abbreviations; the units have international symbols and are not “abbreviations”

Response: Done. We have revised our manuscript accordingly.

Reviewer’s comment (5).

Page 14 - Line 385 Correct “Acknowledgments” to “Acknowledgements” 

Response: Done. We have revised our manuscript accordingly.

 

Thank you for considering once again this manuscript.

Author Response File: Author Response.pdf