Development of Foam-Free Biosurfactant Production Processes Using Bacillus licheniformis
Round 1
Reviewer 1 Report
Comments and Suggestions for Authors
Comments and Suggestions for Authors:
1. Line 175, when measuring the surface tension of a cell-free supernatant, why is it measured at least twice per sample? The mean and standard deviation are calculated by doing at least one replicate experiment per sample under the same culture conditions, not by measuring each sample twice.
2. Line 226, "Yp/x" should be "Yp/s".
3. Line 259, please re-describe the change in curve shown by bacterial growth and glucose consumption.
4. Combine the two graphs in Figures 1-4 into a single graph. How are surface tension, biomass, and pH related in Figure A?
5. What is the direct effect on biosurfactant and foam formation by varying the initial amount of oxygen in the seed cell and bioreactor under oxygen-limited conditions? Please provide more visual data, such as the titer of the biosurfactant, and the change in the level of the bioreactor? Which of the three methods mentioned in the article is better?
6. Please combine the two graphs in Figure 5 into a single graph.
7. Line 485, "Increased glucose concentration does not allow for higher amounts of biosurfactant to be produced", how is this conclusion reached?
8. What is the method for quantifying biosurfactants? The amount of biosurfactant in the medium is not the same when the surface tension is similar. It is inaccurate to judge changes in biosurfactant content by surface tension alone.
9. In section 3.3, the biomass of Bacillus licheniformis EL3 was significantly lower than that of Bacillus subtilis DSM10 and JABs24, but there was no significant difference in the amount of biosurfactant, why?
10. In Table 1, where "Yx/s" and "Yp/s" are both low, "YP/X" and "qBS" are high? What are the specific reasons?
Author Response
Please see the attachment
Author Response File: 
Author Response.pdf
Reviewer 2 Report
Comments and Suggestions for Authors
Dear authors, the manuscript "Development of foam-free biosurfactant production processes using Bacillus licheniformis" is quite interesting and worth investigation:
1- It is well-written and structured,
2- Are you sure the micro-aeration is the best approach? I mean, the yield is extremelly low. In other words, on the one hand, you avoid the foam formation, on the other hand, the yield is too low.
3- Have you considered that the micro-aeration can be correlated to different isorfms?
4- Somehow, you should make clear that there are other strategies related to foam formation, for instance continuous foam recovery, addition of anti foaming (culture medium), etc.
Regards
Author Response
Please see the attachment
Author Response File: Author Response.pdf
Reviewer 3 Report
Comments and Suggestions for Authors
The manuscript by Leal and colleagues describes the characterization and performance of Bacilllus licheniformis in oxygen-limited fermentations using glucose as carbon source. The oxygen-limited condition provides the significant advantage of foam-free biosurfactant production. The produced biosurfactant was Lichenysin, a compound that is very similar in structure and biosurfactant properties to the well-studied surfactin.
The authors conducted multiple fermentation runs, where they compared different conditions (+/- oxygen saturation at the beginning of the run; pre-culture aerobic or oxygen-limited; preculture medium; amount of supplemented glucose). They found that an oxygen saturation at the beginning of the fermentation is beneficial for the production of Lichenysin, was well as a preculture in LB and a glucose amount of 20-30 g turned out optimal.
Unexpectedly, the biosurfactant yields were in the same ranges as compared to similar experiments conducted with Bacillus subtilis, that is less well adapted to oxygen-limited conditions. However, the yield/biomass and the specific productivity were higher than published values.
The presented work is scientifically sound and well described. The results are carefully discussed taking into account similar studies with other B. subtilis species. Although the expected advantage of using B. licheniformis could not be transferred into increased biosurfactant production yields, the results are promising and further improvement in the sense of biomass productivity could potentially yield an improved process.
Author Response
The authors would like to thank the reviewer for the critical evaluation and assessment of our work, for the valuable comments, and for providing us the opportunity of revising the original manuscript.
Reviewer 4 Report
Comments and Suggestions for Authors
The authors have not done any kind of physicochemical characterization like HPLC. LCMS/MS. FTIR or any other kinb of characterization, to claim that the biosurfactant produced is surfactine, or ; lipopeptide; surfactant;.
Comments on the Quality of English Language
The English is fine
Author Response
The authors would like to thank the reviewers for their critical evaluation and assessment of our work, for their valuable comments, and for providing us the opportunity of revising the original manuscript. Please find below the point-by-point answers to your questions and comments.
Reviewer #4
Question 1. The authors have not done any kind of physicochemical characterization like HPLC, LCMS/MS, FTIR or any other kind of characterization, to claim that the biosurfactant produced is surfactin or lipopeptide surfactant.
Answer 1. Thank you for your valuable suggestion. The authors agree that the chemical characterization of the biosurfactant produced by Bacillus licheniformis EL3 would significantly contribute to improve the quality of this work. For that reason, a FTIR analysis of the purified biosurfactant was performed, as well as for commercial surfactin (SIGMA-Aldrich, 99% purity). The results of this characterization are presented in the revised manuscript (lines 225-231; lines 558-572; Figure 6). The FTIR spectrum obtained for the biosurfactant produced by B. licheniformis EL3 (Figure 6A) exhibited the functional groups characteristic of lipopeptide biosurfactants, as discussed in detail in the revised manuscript (lines 558-572), and was almost identical to that obtained for commercial surfactin (Figure 6B). This results indicate, without doubt, that the biosurfactant produced by B. licheniformis EL3 belongs to the surfactins family (that includes surfactin, lichenysin, and pumilacidin).
Considering that lichenysin is the only lipopeptide biosurfactant reported to be produced by B. licheniformis strains (Gudiña EJ, Teixeira JA. 2022. Bacillus licheniformis: The unexplored alternative for the anaerobic production of lipopeptide biosurfactants? Biotechnology Advances 60: 108013. https://doi.org/10.1016/j.biotechadv.2022.108013), we could consider that the biosurfactant produced by B. licheniformis EL3 is lichenysin; however, without further evidence, we decided not to make that statement in the manuscript. However, the FTIR spectrum of this biosurfactant was almost identical to that of commercial surfactin (Figures 6A and 6B). It has to be taken into consideration that lichenysin structure is almost identical to that of surfactin, being the only difference the first amino acid in the peptide ring (glutamic acid or glutamine), which demonstrates that the biosurfactant produced by B. licheniformis EL3 belongs to the surfactin family.
Furthermore, the low surface tension values (29 mN/m) and critical micelle concentration (27 mg/L) obtained with this biosurfactant are in agreement with its lipopeptide nature, according to previous works, as discussed in the revised manuscript.
Round 2
Reviewer 1 Report
Comments and Suggestions for Authors
All the issues were revised, the article can be accepted now.
Author Response
The authors would like to thank the reviewer for the critical evaluation and assessment of our work, for the valuable comments, and for providing us the opportunity of revising the original manuscript.
Reviewer 4 Report
Comments and Suggestions for Authors
The authors need to add more characterization to determine that it is surfactine , LC MS/MS characterization and related chromatographic characterization should be carried out
Comments on the Quality of English Language
The authors need to add more characterization to determine that it is surfactine , LC MS/MS characterization and related chromatographic characterization should be carried out
Author Response
The authors would like to thank the reviewers for their critical evaluation and assessment of our work, for their valuable comments, and for providing us the opportunity of revising the original manuscript. Please find below the point-by-point answers to your questions and comments.
Question 1. The authors need to add more characterization to determine that it is surfactine, LC MS/MS characterization and related chromatographic characterization should be carried out
Answer 1. Thank you for your valuable suggestion. The authors agree that the chemical characterization of the biosurfactant produced by Bacillus licheniformis EL3 would significantly contribute to improve the quality of this work. For that reason, a FTIR analysis of the purified biosurfactant was performed, as well as for commercial surfactin (SIGMA-Aldrich, 99% purity). The results of this characterization are presented in the revised manuscript (lines 225-231; lines 558-572; Figure 6). The FTIR spectrum obtained for the biosurfactant produced by B. licheniformis EL3 (Figure 6A) exhibited the functional groups characteristic of lipopeptide biosurfactants, as discussed in detail in the revised manuscript (lines 558-572), and was almost identical to that obtained for commercial surfactin (Figure 6B). This results indicate, without doubt, that the biosurfactant produced by B. licheniformis EL3 belongs to the surfactins family (that includes surfactin, lichenysin, and pumilacidin).
Considering that lichenysin is the only lipopeptide biosurfactant reported to be produced by B. licheniformis strains (Gudiña EJ, Teixeira JA. 2022. Bacillus licheniformis: The unexplored alternative for the anaerobic production of lipopeptide biosurfactants? Biotechnology Advances 60: 108013. https://doi.org/10.1016/j.biotechadv.2022.108013), we could consider that the biosurfactant produced by B. licheniformis EL3 is lichenysin; however, without further evidence, we decided not to make that statement in the manuscript. However, the FTIR spectrum of this biosurfactant was almost identical to that of commercial surfactin (Figures 6A and 6B). It has to be taken into consideration that lichenysin structure is almost identical to that of surfactin, being the only difference the first amino acid in the peptide ring (glutamic acid or glutamine), which demonstrates that the biosurfactant produced by B. licheniformis EL3 belongs to the surfactin family.
Although a deeper characterization of the biosurfactants herein produced thorough LC-MS could be performed, this is not the objective of the work herein presented. The FTIR results clearly show that the biosurfactant produced by B. licheniformis EL3 is a lipopeptide biosurfactant, belonging to the surfactin family. A deeper chemical characterization through LC-MS would be useful, for instance, to compare the different isoforms produced under the different culture conditions studied, or under aerobic and oxygen-limited conditions, that is, to study the effect of the culture conditions on the lipopeptide isoforms produced by B. licheniformis EL3, which is not the case.
It is relevant to highlight that the objective of the work was to study biosurfactant production in bioreactor under oxygen limited conditions using a B. licheniformis strain, and compare the results obtained in the different approaches used. According to the comments provided by three of the four reviewers that reviewed this manuscript, that objective was achieved and the results proved satisfactory without performing a deeper chemical characterization of the lipopeptide biosurfactant produced. As previously discussed, considering that this a B. licheniformis strain, and the FTIR characterization performed, provides enough information to sustain its lipopeptide nature and fulfills the objectives of the work.
