Investigation on Applying Cyclodextrins in a Fermentation Process for Enhanced Biosurfactant Production by Bacillus licheniformis

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

Reviewer Comments

This manuscript, titled "Investigation on the Application of Cyclodextrins in a Fermentation Process for Enhanced Biosurfactant Production by Bacillus licheniformis," focuses on addressing product inhibition during the fermentation of lichenysin—a biosurfactant derived from Bacillus licheniformis DSM13—through supplementation with α-cyclodextrin (α-CD), β-cyclodextrin (β-CD), γ-cyclodextrin (γ-CD), and dimethyl-β-cyclodextrin (DIMEB). The study assesses the effects of these cyclodextrins with respect to microbial growth, metabolism, and biosurfactant yield, utilizing both small-scale (RTS-1 Personal Bioreactor) and scaled-up (Erlenmeyer shaking flask) cultivation systems, paired with standardized analytical approaches. It exhibits a clear structure and logical flow of reasoning. A key finding—namely, that DIMEB enhances biosurfactant yield by up to 41.43% through mitigating product self-inhibition—holds practical significance for industrial-scale biosurfactant production. However, several issues require clarification or refinement to further enhance the manuscript’s scientific rigor and readability:

 

• The Introduction notes that Bacillus licheniformis produces several lipopeptide biosurfactants, including lichenysin and iturin, yet fails to elaborate on why lichenysin was chosen as the specific target for yield improvement. This gap undermines the rationale behind the study’s focus. It is therefore recommended to supplement information regarding lichenysin’s research value, thereby clarifying the logical basis for the study’s target selection.

 

• Table 1 demonstrates that dimethyl-β-cyclodextrin (DIMEB) at 1.0 g/L significantly reduces biomass (as reflected by OD₈₅₀), while conversely, DIMEB at 2.0 g/L facilitates biomass accumulation. However, the Discussion section fails to account for the mechanism driving this observed concentration-dependent shift in biomass dynamics. Supplementary analysis of this phenomenon is therefore recommended to enhance the clarity of the underlying mechanism.

 

• Section 4. Materials and Methods note the use of a 24-hour-old seed culture to initiate fermentation, yet fails to specify either the seed culture medium (e.g., the complete composition of the LB medium) or the incubation conditions (e.g., temperature, agitation speed). Such details are critical for ensuring experimental reproducibility—consistency in seed culture preparation directly impacts the uniformity of microbial growth kinetics at the fermentation onset, and omitting these parameters could introduce uncontrollable variability into replicate experiments. It is therefore recommended that the seed culture conditions be fully elaborated, including the exact medium composition and incubation parameters, to enable other researchers to accurately replicate the study protocol.

 

• Section 4. Materials and Methods detail the composition of the defined minimal medium but does not specify the purity grade of each reagent. Impurities in low-purity reagents may interfere with Bacillus licheniformis growth or biosurfactant synthesis, affecting experiment reproducibility. It is recommended to supplement the purity grade of all medium components to ensure the reliability of experimental conditions.

 

• Section 5. Conclusions suggests "confirming the presence and characterizing the entrapped biosurfactants" but does not propose specific detection methods. This makes the suggestion overly vague. It is recommended to specify 1-2 feasible experimental methods to enhance the actionability of future research.

Author Response

We appreciate you taking the time to look over our manuscript. We have made changes based on your kind recommendations and have carefully thought about all your suggestions for improving the manuscript. Please see our detailed answer in the below attached file.

Author Response File: Author Response.pdf

Reviewer 2 Report

Comments and Suggestions for Authors

The manuscript presents an interesting study on the use of cyclodextrins to enhance biosurfactant production by Bacillus licheniformis. The topic is timely and relevant, and the experimental results are clearly presented. The work has the potential to make a valuable contribution, but several points could be addressed to further strengthen the manuscript.

 

1. The Abstract could be further enhanced by incorporating key quantitative data and by placing greater emphasis on the novelty of the study.
2. The Introduction notes the ability of cyclodextrins to form inclusion complexes, but does not clearly highlight why they are particularly advantageous for alleviating product inhibition in biosurfactant production. Expanding this rationale would make the study more convincing.
3. The Discussion provides only a limited mechanistic explanation of how cyclodextrins may alleviate product inhibition. Elaborating on why DIMEB, in particular, showed superior performance compared to other cyclodextrins would add clarity and strengthen the impact of the study.
4. The Conclusion effectively summarizes the main findings, but it would be more impactful if it explicitly emphasized the novelty of the study, particularly the distinct advantage of DIMEB compared to other cyclodextrins.

 

Overall, the manuscript is well prepared and of potential interest to the journal readership. With these relatively minor revisions, the clarity and impact of the work would be further enhanced.

Author Response

We appreciate you taking the time to look over our manuscript. We have made changes based on your kind recommendations and have carefully thought about all your suggestions for improving the manuscript. Please see our detailed answers in the below attached file.

Author Response File: Author Response.pdf

Reviewer 3 Report

Comments and Suggestions for Authors

This manuscript investigates the use of cyclodextrins (α-, β-, and γ-CD, as well as methylated derivatives) in fermentation with Bacillus licheniformis to enhance biosurfactant (lichenysin) production. While the idea is relevant to industrial biotechnology, multiple flaws in the study’s design, methodology, and interpretation substantially undermine its scientific validity.

1. The introduction does not adequately establish novelty. Cyclodextrins have been widely applied in biotechnology - for example, as elicitors to enhance metabolite release in plant cell cultures and as solubilizers in pharmaceutical and food processes. They have also been reported to modulate microbial activity in environmental systems. Thus, the general concept of using CDs in bioprocesses is not new. The authors should clarify whether their contribution is specifically the use of Bacillus licheniformis, lichenysin production, or the testing of methylated derivatives.
2. The methods describe "five flasks per condition," but this appears to be technical replication within a single fermentation batch rather than independent biological replicates. Since fermentation is inherently variable, without at least three independent runs, the data cannot support statistical analysis. Nevertheless, ANOVA and Tukey's test were applied, which is invalid in this context. This flaw alone renders the statistical conclusions unreliable.
3. The central claim of increased production by up to 41.43% relies on gravimetric measurements taken after acid precipitation. However, this method is nonspecific and co-precipitates proteins, carbohydrates, and salts from the medium, which inflates yield values. No chromatographic or spectroscopic methods (e.g., HPLC, LC-MS, or NMR) were employed to confirm the identity or purity of the compounds. Although surface tension and emulsification index measurements are reported, these are indirect and cannot substitute for quantitative confirmation. Therefore, the data do not prove increased lipopeptide production.
4. The hypothesis that CDs enhance biosurfactant yield by forming inclusion complexes with lichenysin and reducing product inhibition is speculative and lacks supporting evidence. No experiments were performed to demonstrate complex formation, such as ITC, NMR, or fluorescence. The alternative explanation that CDs directly inhibit growth or alter nutrient availability is more consistent with the data since most CDs reduce the maximum growth rate. However, the discussion does not acknowledge this contradiction.
5. The results show that many CDs, particularly α-CD, inhibit microbial growth. Only one condition, 2 g/L DIMEB, showed a modest increase in final product yield; however, the abstract and conclusion emphasize a broadly "enhancing" effect. This framing is misleading and does not accurately represent the data.
6. Critical micelle concentration (CMC), a standard parameter for biosurfactant characterization, was not determined.
7. Industrial feasibility is not addressed: cyclodextrins are relatively costly additives, and the study does not evaluate whether supplementation at gram-per-liter scale is economically realistic.
8. The rationale for the chosen CD concentrations (0.5, 1.0, 2.0 g/L) is not explained. Possible effects on osmotic pressure or nutrient balance are ignored.

Although the topic is relevant, the study has critical flaws. A complete redesign is required for the work to provide scientifically valid insights. This redesign would entail independent biological replicates, specific analytical validation (HPLC/LC-MS/NMR), direct mechanistic experiments, and realistic industrial evaluation.

Author Response

We appreciate you taking the time to look over our manuscript. We have made changes based on your kind recommendations and have carefully thought about all your suggestions for improving the manuscript. Please see our detailed answers in the below attached file.

Author Response File: Author Response.pdf

Reviewer 4 Report

Comments and Suggestions for Authors

Sakiyo and Nemeth wrote an article on enhancing the production of biosurfactant by a Bacillus strain. The work is valuable, although its quality is low and careless. Below are a few comments that, in my opinion, will help improve the manuscript.
1)    The abstract is rather vague and does not show specific values illustrating the effect of cyclodextrin on biosurfactant production.
2) The Introduction does not clearly indicate a causal relationship between the addition of cyclodextrin and biosurfactant production. What were the reasons for proposing such a hypothesis? 
3)    The quality of the figures is poor, both in terms of legibility and content (no descriptions of the axes with the units used, no uniform number of decimal places, etc.). The legends are unclear and the axes are inconsistent.
4)    Why was OD at 850 nm used to measure growth kinetics instead of the standard 600 nm?
5)    In Figure 2 and its description, it is unclear on what basis such fitting curves were adopted. There is no information on the curve fitting coefficient (R²), which makes it difficult to interpret the results.
6)    In Figure 3 (its description and heading), there are no explanations as to what each graph represents. 
7)    It is not certain whether the compound (or compounds) isolated according to the procedure described in 3.7 is a biosurfactant. These may be other compounds (e.g. small molecules, some oligosaccharides) which themselves reduce surface tension without exhibiting surfactant properties (e.g. amphiphilicity). There is no chemical identification of the nature of the compound(s).
8) The discussion of the results and conclusions are rather vague and, in view of the above comments, too far-reaching.

Author Response

We appreciate you taking the time to look over our manuscript. We have made changes based on your kind recommendations and have carefully thought about all your suggestions for improving the manuscript. Please see our detailed answers in the below attached file.

Author Response File: Author Response.pdf

Round 2

Reviewer 3 Report

Comments and Suggestions for Authors

I would like to thank the authors for their thorough and thoughtful revisions. They addressed the vast majority of my concerns, notably by clarifying the use of independent biological triplicates and improving the framing of the study's novelty and discussion of the proposed mechanism. The authors also included the requested acknowledgment of the limitations of the gravimetric method in the conclusions section. I have no further objections. The manuscript is much improved and provides valuable proof of concept. I recommend acceptance.

Author Response

Authors are really grateful for the respected Reviewer's all effort to improve their manuscript via suggestions and recheck it many times while they reached his/her notes on "I have no further objections."Best regards: Aron Nemeth

Reviewer 4 Report

Comments and Suggestions for Authors

Thank you authors for their kind responses. I feel that the authors improved the manuscript. However, I still feel that the quality of the figures is low. It refers to the bars, legend font (and text of the legend which is unclear) the axis labels etc. Moreover, the style used is incoherent (sometimes is "Time (hour)" sometimes "Time (H)" etc. By the way the abbreviation of hours is h not H.

Author Response

Authors are very grateful for the respected Reviewer and his/her last suggestions focusing on incoherency of Figure axis and labels. Time axis were uniformed in form of Time (h), bar diagram also unified regarding the position of control, clarified "U max" on Fig.1. for Specific growth rate. We hope that our maunscript is now good enough for acceptance for publication. Best regards: Aron Nemeth