Controlling the Formation of Foams in Broth to Promote the Co-Production of Microbial Oil and Exopolysaccharide in Fed-Batch Fermentation

Round 1

Reviewer 1 Report

The manuscript deals with the optimisation of the fermentation process of Sporidiobolus pararoseus for its bitotechnological simultaneous production of oil and EPS. Authors have already published results on this topic confirming their research activity of the research team. The objectives of the study are clearly presented in the introduction part with appropriate bibliography. In order to reduce foaming in culture media authors propose to use anti-foaming agents and optimize the bioreactor device.

Abstract could be improved due to awkward sentence (the first one) and precise the abreviations PPE and CSL.

L.115 : strange to start with Figure 2 why not figure 1 as it is the first appearing in the manuscript.

Concerning the home-made technique used for analyzing defoaming, can authors give a reference for this?

L.128 : Could author precise a little on the corn steep liquor? Supplier? Origin? Way of production ? characterization?

L/136-137 : analysis of 600 nm was used to analyse the concentration of microbial oil and EPS is quite uncommon? Can author giver precisions as generally it is used to monitor cell growth?

 

L. 153 : Authors write “S. pararoseus JD-2 is an aerobic bacterium” isn’t it rather a yeast ? please correct

 

Figure 1 : precise in the legend histograms represente foams and symbols associatecd to measured paramters otherwise it is understandable.

Figure 2 : precise the legend for the colors of histograms and Figure 2.b precise the four defoamers used in the MS part.

 

The results are interesting but should be clearly presented and ordered in order the readers could follow the messages and interpretations of  authors. Please re-write the paper in order it could be more useful for the scientific community. In this state the manuscript could not be published.

Author Response

<Response to reviewer 1#>

Thank you for your useful comments and suggestion on the structure of our manuscript. We have accordingly modified the manuscript (The lines are in red font), and detailed corrections are listed below point by point:

1) Abstract could be improved due to awkward sentence (the first one) and precise the abbreviations PPE and CSL.

• : Sorry for the trouble we brought you. According to your suggestions, we have now rewritten the “Abstract” of this manuscript. In addition, this manuscript has been edited by professional editors at Editage (Shanghai, China), who provides English-improvement services. Therefore, we think the quality of the English is satisfactory for this journal.

2) L.115 : strange to start with Figure 2 why not figure 1 as it is the first appearing in the manuscript.

• : Sorry for the trouble we brought you. According to your suggestions, the order of figures in this manuscript has been changed.

3) Concerning the home-made technique used for analyzing defoaming, can authors give a reference for this?

• : Thank you very much for your constructive advice. The home-made techniques decreased in this manuscript are largely based on previous studies (Tamura, T.; et al., Direct observation of foam film rupture by several types of antifoams using a scanning laser microscope. J Colloid Interface Sci 1999, 213(1), 179-186.). And many researches have been reported that the home-made device used for recycling the foams is beneficent to increase the yield of target products and to improve the cell growth (Anic, I.; et al., Foam adsorption as an ex situ capture step for surfactants produced by fermentation. J Biotechnol 2017, 258, 181-189; Anic, I.; et al., Production of rhamnolipids by integrated foam adsorption in a bioreactor system. AMB Expr 2018, 8, 122; Long, X. W.; et al., Enhanced rhamnolipids production via efficient foam-control using stop valve as a foam breaker. Bioresource Technol 2017, 224, 536-543). Therefore, we think the home-made device in this study can be used to recycle the foam. And the related references have been listed in the main manuscript. We hope you will agree with my explanation and it not affects your decision.

4) L.128 : Could author precise a little on the corn steep liquor? Supplier? Origin? Way of production ? characterization?

• : Sorry for the trouble we brought you due to our mistake. The corn steep liquor was purchased from Shandong Shouguang Juneng Golden Corn Co., Ltd., China. However, the other information, including the origin of corn, the way of production and the components of corn steep liquor, is still unknown. We tried to communicate with the suppliers, but they did not tell us more information. Therefore, we hope you will agree with my explanation and it not affects your decision.

5) L/136-137 : analysis of 600 nm was used to analyse the concentration of microbial oil and EPS is quite uncommon? Can author giver precisions as generally it is used to monitor cell growth?

• : Sorry for the trouble we brought you. The spectrophotometer at 600 nm was used to measure the biomass of samples after an appropriate dilution and not used to analyse the concentration of microbial oil and EPS. The concentration of microbial oil and EPS were detected by weight after extraction (Wang, H.;et al., Co-production of lipid, exopolysaccharide and single-cell protein by Sporidiobolus pararoseus under ammonia nitrogen-limited conditions. Bioprocess Biosyst Eng 2020, 43, (8), 1403-1414; Han, M.; et al., Effects of nitrogen on the lipid and carotenoid accumulation of oleaginous yeast Sporidiobolus pararoseus. Bioproc Biosyst Eng 2016, 39, (9), 1425-1433). For monitoring cell growth, 200 μL of sample was taken from the shake flasks or fermenter every 4 hours. Subsequently, these samples were used to analyze using a spectrophotometer at 600 nm after 25 times dilution. The detail information has been listed in the main manuscript. I hope you will agree with my explanation and it not affects your decision.

6) L. 153 : Authors write “S. pararoseus JD-2 is an aerobic bacterium” isn’t it rather a yeast ? please correct.

• : Sorry for the trouble we brought you, and thank you very much for your direction. S. pararoseus JD-2 is an yeast and an aerobe. The detail information has been changed in the main manuscript.

7) Figure 1 : precise in the legend histograms represent foams and symbols associated to measured paramters otherwise it is understandable.

• : Sorry for the trouble we brought you, and thank you very much for your direction. The legend of figure has been changed, and more information of figures, including histograms and symbols, has been discussed in the main manuscript.

8) Figure 2 : precise the legend for the colors of histograms and Figure 2.b precise the four defoamers used in the MS part.

• : Sorry for the trouble we brought you, and thank you very much for your direction. The legend of figure has been changed, and more information of figures, including histograms and symbols, have been discussed in the main manuscript.

9) The results are interesting but should be clearly presented and ordered in order the readers could follow the messages and interpretations of authors. Please re-write the paper in order it could be more useful for the scientific community. In this state the manuscript could not be published.

• : Thank you for your recognition of our study, and sorry for the trouble we brought you. According to your suggestions, we have now re-write through this manuscript again and we are sure that there have been more rigs here. In addition, this manuscript has been edited by professional editors at Editage (Shanghai, China), who provides English-improvement services. (In red font). We are sure that there are no longer any spelling mistakes. Therefore, I think the quality of the English is satisfactory for this journal.

Finally, we eagerly hope that you can give us you cherish advice. Thank you very much for your attention and consideration. And longing for your replication!

Sincerely yours,

Jian-Zhong Xu

 

 

Reviewer 2 Report

This article contains some significant data and scientific effort however, the English language used throughout the article is very weak, and therefore the whole article needs to be reconstructed and rewritten by a native English language person or a specialist.

I would recommend this paper for publication in the Fermentation journal after a major English editing and a careful consideration of the following comments:

 

1. Change the title to: ‘Controlling the foam formation in broth to promote the co-production of microbial oil and exopolysaccharide in fed-batch fermentation’
2. In the Abstract:

- the word foams should be changed to foam in most cases, this also applies to many places in the manuscript.

- write CSL in full as this is the 1^st time mentioned and do this with all abbreviations.

3. The English language needs revision for grammar and form. There are too many errors and sentences requiring major amendments. The following points are just examples:

• line 33&34: it should be ‘short production time and low pollution to environment’ instead of ‘short production period and little pollution to environment’
• lines 34&35: delete ‘because microbial oil is usually more expensive 35 than vegetable oil’. This part of the sentence brings no meaning and shows poor use of the English language
• Line 37: use ‘strategies have been applied instead of ‘methods have been done’
• Information in lines 44 to 54 can be easily rewritten as one or two sentences with a maximum of 3 lines. The strain’s information and its story is already present in the Materials and Method’s section

4. The introduction should include a paragraph on the different types of antifoams. It can also include a paragraph on unconventional techniques for controlling the foam formation in the fermentation medium such as using seawater. You may refer to those 2 papers: https://doi.org/10.3390/pr9101841 and https://doi.org/10.3390/pr9081399

5. Make sure that ‘S. pararoseus’ and all scientific names are in italic throughout the manuscript.

6. In line 99: you stated that inoculum size is 10% (v/v), please provide more information such as cell concentration or density.

7. In line 109: it should be written 10,000 rpm instead of ‘10,000 r/min’. And you need to convert rpm to g or RCF or at least provide the centrifuge make and model.

8. Define EPS and write in full when first mentioned

9. Please add a sub-section on the antifoam substances that you have used in this study. Full information should be provided.

10. The materials and Method section should contain a sub-section on statical analysis.

11. The Figures’ quality needs major improvement. Convert to colored images, increase resolution, and insert the legend in the graph. The readers will never know what these pars and lines are referring to unless you define that on the Figures!

12. The conclusion should include a statement on the work novelty, the challenges, and limitations faced in the current research, and the perspective for future research.

Author Response

<Response to reviewer 2#>

Thank you for your useful comments and suggestion on the structure of our manuscript. We have accordingly modified the manuscript (The lines are in red font), and detailed corrections are listed below point by point:

1) This article contains some significant data and scientific effort however, the English language used throughout the article is very weak, and therefore the whole article needs to be reconstructed and rewritten by a native English language person or a specialist.

• : Thank you for your recognition of our study, and sorry for the trouble we brought you. According to your suggestions, we have now re-write through this manuscript again and we are sure that there have been more rigs here. In addition, this manuscript has been edited by professional editors at Editage (Shanghai, China), who provides English-improvement services. (In red font). We are sure that there are no longer any spelling mistakes. Therefore, I think the quality of the English is satisfactory for this journal.

2) Change the title to: ‘Controlling the foam formation in broth to promote the co-production of microbial oil and exopolysaccharide in fed-batch fermentation’.

• : Thank you very much for your constructive advice. According to your suggestions, the title of this manuscript has been changed to “Controlling the foam formation in broth to promote the co-production of microbial oil and exopolysaccharide in fed-batch fermentation”.

3) In the Abstract:- the word foams should be changed to foam in most cases, this also applies to many places in the manuscript; - write CSL in full as this is the 1st time mentioned and do this with all abbreviations.

• : Sorry for the trouble we brought you, and thank you very much for your constructive advice. According to your suggestions, we have now rewritten the “Abstract” of this manuscript. In addition, this manuscript has been edited by professional editors at Editage (Shanghai, China), who provides English-improvement services. Therefore, we think the quality of the English is satisfactory for this journal.

4) The English language needs revision for grammar and form. There are too many errors and sentences requiring major amendments. The following points are just examples:line 33&34: it should be ‘short production time and low pollution to environment’ instead of ‘short production period and little pollution to environment’lines 34&35: delete ‘because microbial oil is usually more expensive 35 than vegetable oil’. This part of the sentence brings no meaning and shows poor use of the English languageLine 37: use ‘strategies have been applied instead of ‘methods have been done’Information in lines 44 to 54 can be easily rewritten as one or two sentences with a maximum of 3 lines. The strain’s information and its story is already present in the Materials and Method’s section.

• : Sorry for the trouble we brought you. According to your suggestions, we have now re-write through this manuscript again and we are sure that there have been more rigs here. In addition, this manuscript has been edited by professional editors at Editage (Shanghai, China), who provides English-improvement services. (In red font). We are sure that there are no longer any spelling mistakes. Therefore, I think the quality of the English is satisfactory for this journal.

5) The introduction should include a paragraph on the different types of antifoams. It can also include a paragraph on unconventional techniques for controlling the foam formation in the fermentation medium such as using seawater. You may refer to those 2 papers: https://doi.org/10.3390/pr9101841 and https://doi.org/10.3390/pr9081399.

• : Thank you very much for your constructive advice. According to your suggestions, we have discussed the other antifoams strategies, for example using seawater to control the foam formation, and the related references (i.e., https://doi.org/10.3390/pr9101841 and https://doi.org/10.3390/pr9081399) have been added in the main manuscript. Given the space of this manuscript and the highlights of this manuscript, however, we did not discussed the different types of antifoams in a new paragraph. We hope you will agree with my explanation and it not affects your decision.

6) L. 153 : Authors write “S. pararoseus JD-2 is an aerobic bacterium” isn’t it rather a yeast ? please correct.

• : Sorry for the trouble we brought you, and thank you very much for your direction. S. pararoseus JD-2 is an yeast and an aerobe. The detail information has been changed in the main manuscript.

7) Make sure that ‘S. pararoseus’ and all scientific names are in italic throughout the manuscript.

• : Thank you for your reminding. We have now read through this manuscript again and we are sure that all scientific names are in italic throughout the manuscript.

8) In line 99: you stated that inoculum size is 10% (v/v), please provide more information such as cell concentration or density.

• : Sorry for the trouble we brought you, and thank you very much for your constructive advice. The cell in logarithmic phase was used as the inoculum, which was obtained from a seed culture for cultivating about 10 h. The related information has been listed in the main manuscript.

9) In line 109: it should be written 10,000 rpm instead of ‘10,000 r/min’. And you need to convert rpm to g or RCF or at least provide the centrifuge make and model.

• : Thank you for your reminding and constructive advice. We have provided the centrifuge make and model in the main manuscript.

10) Define EPS and write in full when first mentioned.

• : Sorry for the trouble we brought you. According to your suggestions, we have written in full name of EPS.

11) Please add a sub-section on the antifoam substances that you have used in this study. Full information should be provided.

• : Thank you for your reminding and constructive advice. The antifoams used in this study are all commercial products. For example, PPE is the polyoxyethylene polyoxypropylene pentaerythritol ether, GPE is the polyoxypropylene oxyethylene glycol ether, DSA-5 is the defoamer for bean products. The soybean oil was purchased from Wilmar International (China) Limited. The related information has been listed in the main manuscript. However, we did not add a sub-section on the antifoam substances. We hope you will agree with my explanation and it not affects your decision.

12) The materials and Method section should contain a sub-section on statical analysis.

• : Thank you for your reminding and constructive advice. According to your suggestions, we have provided the sub-section on statistical analysis in the main manuscript.

13) The Figures’ quality needs major improvement. Convert to colored images, increase resolution, and insert the legend in the graph. The readers will never know what these pars and lines are referring to unless you define that on the Figures!

• : Thank you for your reminding and constructive advice. We have changed the information of figures in the main manuscript, including the legends of the figures and the detail information of the symbols in the figures.

14) The conclusion should include a statement on the work novelty, the challenges, and limitations faced in the current research, and the perspective for future research.

• : Thank you for your reminding and constructive advice. According to your suggestions, we have now re-write through this manuscript again and we are sure that there have been more rigs here.

Finally, we eagerly hope that you can give us you cherish advice. Thank you very much for your attention and consideration. And longing for your replication!

Sincerely yours,

Jian-Zhong Xu

 

 

Reviewer 3 Report

Overall impressions

The manuscript is well structured. The experimental design is consistent with the results, which support the conclusion. However, the control of foam formation is already a component of any process optimization, and therefore I found low originality and significance in this work.

Minor corrections

Figure 2. Quality of the Panel A must be improved

Lines 296 and 304: Scientific name should be written with italics: S. pararoseus

Author Response

<Response to reviewer 3#>

Thank you for your useful comments and suggestion on the structure of our manuscript. We have accordingly modified the manuscript (The lines are in red font), and detailed corrections are listed below point by point:

1) The manuscript is well structured. The experimental design is consistent with the results, which support the conclusion. However, the control of foam formation is already a component of any process optimization, and therefore I found low originality and significance in this work.

• : Thank you for your recognition of our study and for your constructive advice. You are right, this paper mainly focus on controlling the foam formation. To do this, we discussed the relationships between foams and the key factors involved in foaming, and used different strategies to control the foams formation in fed-batch fermentation by S. pararoseus JD-2, including screening of defoamers, optimization of adding ways of CSL as well as utilization use of foams backflow devices. Based on the description reported by Junker, foaming is considered a “general nuisance” in industrial fermentation because fermentation process provides the essential conditions for foam formation (Junker, B., Foam and its mitigation in fermentation systems. Biotechnol Progr 2007, 23, (4), 767-784.). Therefore, we think that our results reported in this study could be used as technical reference for producing the frothing products in fed-batch fermentation. We hope you will agree with my explanation and it not affects your decision.

2) Figure 2. Quality of the Panel A must be improved.

• : Thank you very much for your constructive advice. According to your suggestions, Figure 2 has been revised.

3) Lines 296 and 304: Scientific name should be written with italics: S. pararoseus.

• : Sorry for the trouble we brought you, and thank you very much for your constructive advice. According to your suggestions, we have now rewritten the “S. pararoseus” with italics. In addition, we have now read through this manuscript again and we are sure that all scientific names are in italic throughout the manuscript.

Finally, we eagerly hope that you can give us you cherish advice. Thank you very much for your attention and consideration. And longing for your replication!

Sincerely yours,

Jian-Zhong Xu

 

 

Round 2

Reviewer 1 Report

Authors have improved the content of the manuscript. Figures are clearer, the content has been proof-edited. Additional references have been mentioned. MS has been improved too. This revised manuscript could be published in state.

Author Response

Thank you for your useful comments and suggestion on our manuscript and Thank you for your affirmation of our study.

Reviewer 2 Report

The manuscript has significantly improved compared to the original however, I strongly recommend a second English revision as some minor errors still exist. In addition, the quality of the Figures is still quite poor, I strongly recommend improving the resolution and converting to color instead of black and white, this will significantly enhance the clarity. 

Author Response

1)     The manuscript has significantly improved compared to the original however, I strongly recommend a second English revision as some minor errors still exist. In addition, the quality of the Figures is still quite poor, I strongly recommend improving the resolution and converting to color instead of black and white, this will significantly enhance the clarity.

Rep.: Thank you for your affirmation of our study and thank you very much for your constructive advice. According to your suggestions, we have now read through this manuscript again and we have revised the mistakes in the manuscript. In addition, you are right, the figures with color are easier to distinguish. I would love to change the figures with colors, but I have no the original data of these figures at now because they are stored in office computer. As you're aware, we are on holiday now in China. I hope you will agree with my explanation and it not affects your decision.