Optimization of Bacterial Cellulose Production from Waste Figs by Komagataeibacter xylinus
Round 1
Reviewer 1 Report
Comments and Suggestions for AuthorsThe work fermentation-3151879 is devoted to a relevant topic, namely, to the biosynthesis of bacterial cellulose from food production waste. The positive aspects of the work include the organization of work taking into account the world experience described in the literature and the use of well-described and generally accepted techniques, equipment, and analysis methods. Therefore, the results seem reliable.
The work has a number of serious shortcomings:
1) The concept of fig processing is criticized, since the authors themselves write in the introduction that fig waste can be used to obtain popular food products: jam, marmalade, pastilles, jam, etc. Thus, obtaining bacterial cellulose from this type of raw material competes with food production.
The justification should indicate what percentage of figs is lost during processing. If it is proposed to process figs damaged by microbial spoilage, then, since this is a sugary product, numerous groups of microorganisms, including pathogenic ones, can develop on it, then the feasibility of such processing is very questionable. The justification should be strengthened. It is not clear what kind of fig waste is proposed to be used.
2) The work is trivial. Sugary raw materials are used, a known producer is used, the search for optimal conditions for a specific raw material and producer is an applied production problem. What is the novelty of the work?
3) In paragraphs 3.4 and 3.6, such an indicator as the yield of bacterial cellulose should be added. Make calculations according to Hestrin S., Schramm M. Synthesis of cellulose by Acetobacter xylinum: II. Preparation of freeze-dried cells capable of polymerizing glucose to cellulose // Journal of Biochemistry. –1954. – Vol. 58, N 2. – P. 345-352. https://doi.org/10.1042/bj0580345
If the authors claim to implement the technology, then a very important technological parameter is the yield of bacterial cellulose, calculated from the concentration of sugars in the medium. According to Fig. 1,c, with a sugar concentration in the medium of 20 g / l, the authors obtain a yield of bacterial cellulose of about 33%, and with an increase in the concentration of sugars in the medium to 60 g / l, the authors obtain a yield of bacterial cellulose of about 13%, with an increase in the concentration of sugars in the medium to 80 g / l, the authors obtain a yield of bacterial cellulose of about 7.5%. Thus, with an increase in the concentration of sugars in the medium, the efficiency of their use by the producer for the biosynthesis of bacterial cellulose decreases.
I believe that in paragraph 3.6, the wrong parameter was chosen; it was necessary to strive not for an increase in the productivity of bacterial cellulose (g / l), but for an increase in the yield of bacterial cellulose from the concentration of sugars in the medium (%).
4) Some paragraphs are redundant. It is not clear why it was necessary to use a central composite design? There are optimal pH and temperature levels for the strain and they do not change with a change in the concentration of sugars, since they are determined by the biological characteristics of the producer. This is obvious and this has been confirmed as a result of this work.
5) Paragraph 2.5 Why was this washing method chosen? It is known that at 80 C, mercerization of bacterial cellulose in an alkali solution can begin.
6) In paragraph 3.4, the comparison of experimental data and literary data is incorrect. For example, the authors say that their work yielded more bacterial cellulose than [12]. Do the authors think that 8.4 g/l is more than 10.8 g/l? The authors do not cite a large body of work where the productivity of bacterial cellulose was much higher than in the authors' works.
For example, in the work
In the work of Fan X., Gao Y., He W., Hu H., Tian M., Wang K., Pan S. Production of nano bacterial cellulose from beverage industrial waste of citrus peel and pomace using Komagataeibacter xylinus // Carbohydr Polym. - 2016. - No. 151 . -P. 1068–1072. https://doi.org/10.1016/j.carbpol.2016.06.062 the productivity of bacterial cellulose was 13.7–14.2 g/l;
In Guo X., Chen L., Tang J., Jo¨nsson L.J., Hong F.F. Production of bacterial nanocellulose and enzyme from [AMIM] Cl pretreated cotton waste fabrics: effects of dyes on enzymatic saccharification and nanocellulose production // J Chem Technol Biotechnol. – 2016. – No. 91(5). – R. 1413–1421. https://doi.org/10.1002/jctb.4738 bacterial cellulose productivity was 86.7 g/l;
In Kuo C.H., Huang C.Y., Shieh C.J., Wang H.M.D., Tseng C.Y. Hydrolysis of orange peel with cellulase and pectinase to produce bacterial cellulose using Gluconacetobacter xylinus // Waste Biomass Valorization. – 2017. –№ 10. – R. 1-9. https://doi.org/10.1007/s12649-017-0034-7 bacterial cellulose productivity was 37.88-40.19 g/L;
In Dubey S, Singh J, Singh RP Biotransformation of sweet lime pulp waste into high-quality nanocellulose with an excellent productivity using Komagataeibacter europaeus SGP37 under static intermittent fed-batch cultivation // Bioresour Technol. – 2018. – Vol. 247. – R. 73–80. https://doi.org/10.1016/j.biortech.2017.09.089 bacterial cellulose productivity was 80.04-82.54 g/L;
7) In all the examples given, the productivity of bacterial cellulose is much higher than in the authors' work and in those works with which they compare their results. Therefore, it is necessary to add the given examples to the discussion.
8) Note on Figure 5. The morphology of the bacterial cellulose samples obtained on the synthetic medium and on the fig medium is very different, but the authors write that they do not differ. This needs to be corrected. What do the elongated scaly plates represent for the bacterial cellulose sample obtained on the fig medium?
Author Response
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Author Response.pdf
Reviewer 2 Report
Comments and Suggestions for AuthorsThis study explores using waste figs as an alternative substrate for producing bacterial cellulose (BC) by Komagataeibacter xylinus. The process was optimized using Plackett-Burman design and response surface methodology. The results showed that Yeast extract significantly impacted BC production, achieving a maximum yield of 8.45 g/L with a dense, ribbon-like structure with 30-49 nm diameters and 70% crystallinity. This research demonstrates that waste figs are an effective substrate for BC production, marking the first detailed study in this area. The study is interesting and reports useful findings for low-cost BC production; however, there are few issues which need to be addressed before publication.
1. Abstract: It is stated that initial sugar concentration was 62.75 g/L, while HS medium contains 20 g/L glucose as the carbon source. How the comparison of two media is justifiable?
2. Introduction needs extensive revision.
- For example, authors need to list some well-known bacterial strains for BC production at the start, as well as describe bio-cellulose synthesis by the cell-free enzyme system. This basic information as well as advancements in cellulose synthesis would give useful information to the readers.
- While this study is mainly focused to produce low-cost BC by using figs waste, it is recommended to briefly discuss the economic analysis of BC production. Authors may refer to review by Kamal et al. (Cost-effective synthesis of bacterial cellulose and its applications in the food and environmental sectors. Gels, 8:552, 2022) and other reviews where this is discussed.
- Line 34: change to ‘similar chemical properties’
- Lines 35-36: hydrophilicity is written twice in same sentence.
- Lines 38-39: Provide specific reference for each mentioned application rather providing references at the end of the sentence. Additionally, separate the applications by comma (,) and extend the list to more advances applications such as using bacterial cellulose as a matrix for phage immobilization for biosensing as well as use of BC in 3D bioprinting.
- Lines 51-60: Provide specific reference for each mentioned waste/supplement. It is recommended to specify waste and supplement used as the carbon sources for BC production as both have different impact on the overall production cost.
- Line 82: Change to ‘it is’.
- Lines 98-100: It is too early to discuss the findings of the study. This last passage of the Introduction should briefly describe the objectives of the study and approaches used. The last two sentences are fine.
3. Materials and methods
- Line 109: correct as 2 wt.%
- Line 110: change to ‘freshly prepared medium’
- Change minutes to ‘min’ throughout the text.
- Unify 4 °C/4°C, ax example. Correct it throughout the manuscript.
- Lines 143-144: Usually the initial pH of the HS medium is acidic due to the presence of citric acid. So, alkali may be used to adjust its pH to 5.0. Please check.
4. Results and discussion
- Figure 5. While SEM image of BC produced by HS medium shows typical fibrous morphology, SEM image of BC produced in medium supplemented with figs extract contains some flakes-like structures. Are these some medium remaining or cellular lysates? Did you wash BC produced in figs extract supplemented medium before SEM analysis?
- Unify Figure/Fig. throughout the text.
- Figure 6. FTIR spectrum of BC produced in HS medium should be provided for comparison.
- Figure 7. The reported crystallinity values look smaller than actual and need to be recalculated considering the area under the peaks. What the small peaks at around 2θ = 27 and 39 represent in Figure 7b? Merge both figures into a single graph for better comparison of peaks.
5. Appropriate use of terminologies is required, such as change
- Concentration of BC to ‘yield of BC’
- Waste fix extract as substrate to ‘waste fix extract as supplement’
- Line 18: microfibrils to ‘nanofibrils’ considering the diameter in nm range
- Medium/media
6. All abbreviations should be defined at first appearance and then used consistently. For example, bacterial cellulose is defined as BC in line 9 but then full form is available at several places such as at lines 20, 22, 23 in the abstract. Check the same issue in the main text.
Comments on the Quality of English LanguageModerate language editing is recommended.
Author Response
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Author Response File: Author Response.pdf
Reviewer 3 Report
Comments and Suggestions for AuthorsThe manuscript by Yilmaz and Goksungur reports BC production by utilizing figs extract as a low-cost carbon source. BC production was comparatively analyzed from HS medium and figs extract supplemented medium as well as through SEM, FTIR, and XRD analyses. The manuscript should be revised according to below comments.
· How did you measure the crystallinity? The relevant details should be provided.
· FTIR spectrum of BC produced by the figs extract medium should be compared with that produced in HS medium.
· SEM images of both BCs are not matching in their fibrous morphology. Some irregular shape particles appear in the SEM image of BC produce by the figs extract medium.
· How did you measure the fiber diameter? Provide relevant details.
· Abstract should contain more results rather providing too much experimental details.
· Objectives of the study should be clarified in the Introduction section to emphasize the novelty.
· All equations should be numbered.
· Table captions are too brief. For example, caption of Table at line 162 and that of Table 2 at lines 185 are too brief and do not provide any details of what is presented in the tables. The same for caption of Table 4.
The produced BC by the figs extract medium should be characterized for physiological properties such as water holding and release properties as well as mechanical and thermal properties.
Comments on the Quality of English LanguageModerate
Author Response
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Author Response File: Author Response.pdf
Round 2
Reviewer 1 Report
Comments and Suggestions for AuthorsThe authors responded to a number of comments, but did not correct the fundamental errors.
1) The authors' response to comment 3:
“The substrate conversion percentages (g bacterial cellulose/g initial sugar × 100) of 32.00%, 17.45%, 12.50% and 7.83% were obtained for 20, 40, 60 and 80 g/L initial sugar concentrations, respectively. It was observed that as the concentration of sugars in the medium increased, the efficiency of the microorganism to utilize them for bacterial cellulose biosynthesis decreased.”
Then the yield of bacterial cellulose should be given in Table 2, not productivity, and this parameter should be used for compositional planning and the results should be reflected in Figure 1, in Section 3.6 and in the conclusions.
2) The authors do not sufficiently discuss the fact that there are works in which much more bacterial cellulose was obtained than in their work. If the authors do not wish to increase the number of links, they should be replaced. The reader should not be misled.
Author Response
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Author Response File: Author Response.pdf
Reviewer 2 Report
Comments and Suggestions for AuthorsWhile the authors responded to all comments, they failed to provide appropriate answers and make corresponding changes in the manuscript in response to some comments. Some inappropriately addressed comments are listed below.
1. Comments 1: A comparison of BC production by using two different media (figs and HS medium) can only be done if both have same amount of initial glucose. Further, a high amount of sugar (glucose) usually have inhibitory effect on the growth of bacteria. Please check relevant literature. Therefore, authors need to reconsider the amount of glucose used in the medium for BC production and making subsequent comparison for BC production.
2. Comments 2:
· Considering the aim of the study to show improved BC production, the authors should be aware that different bacterial strains have varying ability of BC production. Likewise, studies, such as the development of cell-free enzyme systems’ have been carried out for improved BC production. Therefore, it is indeed needed to mention, though briefly, some well-known BC-producing strains and the cell-free enzyme systems to provide readers a clear picture of the research trends in BC production before they read the important findings of the study.
· The reviewer never asked to do economic analysis of BC production, but asked to briefly discuss the economic analysis of BC production. This is again in line with the aim of the study to cost-effectively produce BC and will provide a clear picture of research trends to the readers.
· The cited references 2 and 3 are research article and they are focused on BC production and not the mentioned applications in the manuscript. Again, the authors failed to provide a rational answer and did not make appropriate amendments in the manuscript.
3. Comments 14: I wonder how the pH of the medium containing citric acid was higher than 5 (i.e., more towards alkaline) and the authors needed to add alkali to increase the pH and adjust to 5?
4. Comments 15: Figure 5. It seems that authors have very little knowledge of the fibrous morphology of BC. While authors referred to a non-English publication (Lektemür Alpan et al., 2022), thousands of publications on BC show a clear fibrous morphology (as presented in Figure 5a). It appears that the flakes-like structures in Figure 5b are residuals, probably of figs wastes, which might have remained due to improper washing of BC after harvesting. The authors argument that washing of thick BC sheets becomes challenging does not make much sense as they have only presented the surface morphology of BC in Figure 5b.
5. What does ‘Abs’ show in Y-axis in Figure 6? Merge both Figure 6a and b for better comparison of functional groups. Improve figure quality.
6. Comments 18: Still inappropriate presentation of figure 7a and b. Merging them will provide a better comparison of peaks and visible appearance of peak area. Improve figure quality.
7. Comments 19: Yield is a more appropriate way of estimating the amount of cellulose produced from waste figs. Concentration is generally, though not always, used for solutions, while BC is insoluble in medium or water. Additionally, the comparative potential of a substrate to produce BC should be presented in terms of yield. The authors may refer to literature on BC production from various wastes.
Comments on the Quality of English LanguageA minor language editing is needed.
Author Response
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Author Response File: Author Response.pdf
Reviewer 3 Report
Comments and Suggestions for AuthorsThe response to my comments and revised manuscript shows that authors have only addressed a few comments, leaving unaddressed the others. Some are listed below:
1. Provide all details (including formula) of determining crystallinity in the experimental sections.
2. The current SEM images of BC are not acceptable. The irregular-shaped particles are uncommon in BC morphology. These particles are either figs particles or cellular lysate, caused by the improper washing of BC after harvesting.
3. The experimental details for determining fiber diameter should be mentioned in the experimental section.
4. It is inappropriate to say that characterizing BC produced from figs medium for water holding and release properties, and mechanical and thermal properties is not the scope of this study. It is always important to characterize BC obtained from a new medium for such properties to ensure that it demonstrates superior or at least acceptable properties. Such data should be added to the revised manuscript.
Comments on the Quality of English LanguageMinor language editing
Author Response
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Author Response File: Author Response.pdf
Round 3
Reviewer 1 Report
Comments and Suggestions for AuthorsDear authors!
I agree with you that there is a misunderstanding in terminology.
I will try to explain my idea. You cite the concentration of bacterial nanocellulose, grams/liter, this is indeed a common practice in the world.
However, you set the task of optimization. In this case, you have incorrectly set the optimality criterion. You spend more and more sugar, while the concentration of bacterial cellulose grows in grams/liter, but the product yield, expressed as a percentage of the mass of raw materials, falls! If the product yield falls, then the conditions are moving away from optimal.
The biofactory will not increase the standards for laying raw materials in order to achieve a decrease in the yield of the finished product.
Please change the optimality criterion.
The works you cite contain a methodological error.
Author Response
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Author Response File: Author Response.pdf
Reviewer 2 Report
Comments and Suggestions for AuthorsIt appears that authors have now carefully revised the manuscript; therefore, I am pleased to recommend its acceptance for publication.
Comments on the Quality of English LanguageMinor language editing is needed.
Author Response
Thank you very much for your critics and recommending the acceptance of the manuscript for publication.
Reviewer 3 Report
Comments and Suggestions for AuthorsAccept
Comments on the Quality of English LanguageMinor
Author Response
Thank you very much for your critics and recommending the acceptance of the manuscript for publication.
