Initial Analysis on the Characteristics and Synthesis of Exopolysaccharides from Sclerotium rolfsii with Different Sugars as Carbon Sources
, Yu-Xiang Jia 1, Yan Su 1, Xiao-Yu Zhang 1, Lin-Na Tu 1, Zhi-Qiang Nie 2, Yu Zheng 1,* and Min Wang 1,*Round 1
Reviewer 1 Report
The manuscript improved by the corrections made and is much more comprehensive now. But still, the English language must be checked carefully in the whole manuscript. Also the term bacteria must be removed when talking about S. rolfsii, which is a fungi.
Author Response
Thank you for your kind suggestion on our paper. We are sorry that there were some mistakes in the original manuscript. The term “bacteria” was changed to “mycelium”. (page 2, line 81)
At the same time, we have made some revision through the paper to make it more persuasive and the revisions in the text are highlighted in red.
We have polished the paper in https://www.shinewrite.com/s4.html. We hope that the language is now acceptable for the next review process.
Reviewer 2 Report
In this manuscript entitled “Initial Analysis on the Characteristics and Synthesis of Exopolysaccharides from Sclerotium rolfsii with Different Sugars as Carbon Sources”, the authors investigate the influence of different carbon sources on the properties and synthesis of exopolysaccharides (EPS). They analyzed the differences in viscosity, yield, microscopic morphology and metabolic processes of the EPS of S. rolfsii fermented with glucose, sucrose, and lactose as carbon sources. Their structures were preliminarily characterized only by infrared spectroscopy.
The major criticism of this manuscript is the lack of relevant content for the scientific community and sufficient novelty of the work. The authors only performed an “Initial Analysis on the Characteristics and Synthesis of Exopolysaccharides” (as mention in the title) and “their structures were characterized preliminarily” (as mention in the conclusions). The characterization of EPS only by IR is not sufficient.
In my opinion, the results presented in the manuscript did not “provide a certain reference for explaining the effects of carbon sources on microbial fermentation and widen the horizon for development of new polysaccharide products.”, as said in Lines 317-318. The achievements and importance of the results are not properly presented and discussed. It is not obvious how useful are the results presented! The conclusion looks
like the abstract. In my opinion, the EPS from the different carbon sources need to be fully characterized by other spectroscopic techniques to better understand their structural differences. The authors said in Lines 315-317 “…there is an urgent need for more research leading to a better understanding of their metabolic difference, as well as biosynthesis.” In my opinion, that research should have been done and presented in this manuscript.
Moreover, the manuscript should undergo extensive English revisions and it needs to be careful reviewed of the construction of sentences. It is a constant throughout the manuscript as it is exemplified in the 2nd sentence of Abstract “To investigate the influence of different carbon sources on the properties and synthesis of exopolysaccharides(EPS), three EPSs(GEPS, LEPS and SEPS) were selected from different carbon sources, respectively.”; Lines 25-26: “There are minimal differences were observed…”; Lines 95-96: The micro-structures of the three EPSs were observed by scanning electron microscopy (SEM) (SU1510, Hitachi HighTechnologies, Japan) was used.”; Lines 96-97: “…the EPSs were grounded in a mortar.And then coated…”; Line 213: “…there may be more active groups in SEPS and LEPS, which make it…”
Additionally, there are some writing errors in the manuscript, such as "…K, Na, Ca, Mg, and Fe…" in Lines 206-207, “identifified” in Line 265, “signifificantly” in Line 282, etc. Authors need to make comprehensive corrections to the manuscript.
Another error that appears regularly is the absence of spaces between words. Example: Lines 18-19: “…exopolysaccharides(EPS), three EPSs(GEPS, LEPS and SEPS)…”, etc
Another review of the manuscript that should be carefully done is related to the acronyms. Once a given acronym is mentioned for the first time, it should always be used. The authors must confirm the acronyms in all manuscript.
One important acronym of this paper is EPS. In abstract the authors said: “exopolysaccharides(EPS)” in Line 18; but, in Introduction they said “exopolysaccharide also known as scleroglucan is an extracellular polysaccharide (EPS)”!
The meaning of some acronyms such as GEPS, LEPS, SEPS, etc, must be included.
The units must be uniformed (mL·min–1 or mL/min, h or hour, etc, not both) as well as the decimal places “(FC) >1.3 or <0.77” in Line 147, “2.58 g/L an 2.3 g/L” in Line 163, etc
Line 219: “3.4. FT-IR spectra analysis” The authors said several times “peak” referring to a band. This basic mistake is inadmissible for those working in this area! Additionally, Lines 220-231 must be deleted because is basic information! The IV data needs to be revised. For example: “The absence of peak at 1,726 cm−1 (carboxyl group)…” Line 234. Carboxyl group?! C=O or C-O or O-H?
In some sections, the discussion is poorly written and not well founded. For example:
Line 180: The authors said: “…on the basis of ensuring that polysaccharides have a certain viscosity, we selected SEPS, LEPS and GEPS for further study.” Why didn't they choose the maltose with the best viscosity results?
Line 249-250: The authors said: “GEPS had a higher percentage area (92%) and was considered that there is only one component, and the other component could be ignored”. The authors have to better justify this exclusion since in the chromatogram is obvious the presence of a second peak (corresponding to 8%?).
For all these reasons, I cannot recommend publication in Polymers.
Author Response
Please see the attachment.
Author Response File: 
Author Response.docx
Round 2
Reviewer 2 Report
After the changes and improvements performed by the authors, regarding the discussion of results as well as the inclusion of additional characterization data, I consider the manuscript “Initial Analysis on the Characteristics and Synthesis of Exopolysaccharides from Sclerotium rolfsii with Different Sugars as Carbon Sources” suitable for publication in Polymers after minor revision. A comprehensive English revision must be considered before the manuscript can be accepted.
Author Response
Thank you for your kind suggestion on our paper. The manuscript has been carefully revised considering about the language and grammar problems by Tu Linna from HKUST. We have made some improvement, and the corresponding revisions of manuscript were highlighted in red again. We hope that the language is now acceptable for the next review process.
This manuscript is a resubmission of an earlier submission. The following is a list of the peer review reports and author responses from that submission.
Round 1
Reviewer 1 Report
The most significant proposal in this paper [lines 223-224] is that SEPS is an α-linked glucan. As scleroglucan is defined as a glucan with a β1-3-linked mainchain and regular β1-6-linked sidechains, SEPS is not a scleroglucan. Essentially, the claim is that, when grown on sucrose, the organism shuts down scleroglucan synthesis and starts production of a completely different exopolysaccharide. This novel claim is inconsistent with previously published data [Ref. 8 in this paper, for example], and is not even mentioned in the Abstract. The only structural evidence for this claim is the assignment of a single peak in the FT-IR spectrum. I find this difficult to believe, and such an assertion should be backed up with considerably more structural data.
The authors fail to report some key structural data which many would find important, such as the molecular weights of the various glucans. As Castillo et al. say, "[scleroglucans] may show different branching frequency, side-chain length, and/or molecular weight depending on the producing strain or culture conditions], but the authors have determined none of these parameters [N Castillo et al., Frontiers in Microbiology, 2015, 6, article 1106].
The authors used proteomics approaches to try to map biosynthetic pathways, but the rationale for linking these findings with the scleroglucan characterisation data. For example, lines 247-248, they say "The down regulation of these pathways ………. As well as the polymerisation of EPS [33]". Reference 33 refers to the influence of blocked glycolysis on the growth of human HeLa cells, and I fail to see how a connection can be drawn with microbial production of an exopolysaccharide. Also, lines 250-251, they say "The α-glucosidase, β-glucosidase ….. may result in the low molecular weight of LEPS and reducing its rheological properties". The authors never report the determination of the molecular weights of these samples, and so the reader doesn't know if LEPS does indeed have a lower molecular weight. Since scleroglucan contains only β-linked Glc, then it is not clear how the presence of an a-glucosidase could affect it. In both these cases the authors hypothesis that a change observed in the proteomic study "may result in" structural changes in the scleroglucan or the a-linked EPS SEPS. But they do not provide any convincing rationale for drawing those conclusions.
The authors report that analyses were performed in triplicate, but apparently on the same sample of each EPS. Variability in the characteristics of the samples produced is, in my opinion, more important than analytical variability in analysing a single sample.
If finally accepted, this manuscript would benefit from final editing by a native speaker of English: at times the phrasing is odd, but not enough to hinder understanding.
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Lines |
Comment |
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61-62 |
Should this be OXOID, not DXOID? |
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70 |
What is PDA medium? |
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89-90 |
How was the solution concentrated? By partial evaporation? By use of membrane filters? Or dissolution of lyophilised material? |
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Table 1 |
I read this to mean that a single peak was observed in the sugar analysis, with no minor or trace component sugars. If this is indeed the result, then it should be explicitly stated. If minor monosaccharide components were observed, they should be listed and quantified. In my experience, removal of yeast mannans [present in yeast extract in the culture medium] from microbial polysaccharides is difficult, and mannan contamination common. |
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185-189 |
Protocols not mentioned in the Experimental Section |
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Table 3 |
These data look like replicate analyses on a single sample, but a much more significant result would be on analyses of multiple samples. The cited error ranges reflect analytical method uncertainty, not sample variability. Please provide analytical data for multiple independent samples. |
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212-213 |
Relevance of information on galactose, as the scleroglucan is reported as being purely composed of glucose |
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217-218 |
If they need to use FT-IR to suggest the lack of uronic acids, then they are using hydrolysis conditions unsuitable for showing this in the sugar analysis in Table 1. That data should be reported as neutral sugars. |
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217-220 |
The authors cite the lack of a peak at 1726 cm-1 as evidence for the lack of uronic acids (ie. carboxyl groups), and the presence of a peak at 1650 cm-1 for the presence of carboxyls and/or C=O groups. I find this inconsistent and fail to understand the implications. The authors should clarify their interpretation of the data in consistent terms. Do they suspect the presence of N-acetylated aminosugars? The authors report peaks consistent with C-O-C and C-OH in SEPS, but that substructure is a fundamental part of the sugar ring. It would have to be in all polysaccharides. The authors should more precisely clarify their interpretation of the data. |
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Ref. 17 |
This reference refers to HPLC-ESI MS of labelled monosaccharides, whilst the current paper appears to analyse underivatised monosaccharide. The authors should clarify. |
Reviewer 2 Report
Dear authors,
the manuscript entitled "Initial Analysis on the Characteristics and Synthesis
of Exopolysaccharides from Sclerotium rolfsii with Different Sugars as Carbon Sources" is of general interest for the readers of Polymers. In sum enough experiments were carried out and some conlusions were drawn from the results. However, the overall structure and the material and methods part must be rewritten to make it clear to follow, and to allow the reader to get all necessary information. Also some conclusions must be evaluated carefully, and the correlation of results and conclusions must be reworked. The Englisch language must be improved to make it a straight manuscript. Some current references are missing, which might help to improve the structure of the manuscript.
Concerns:
Abstract: No abbreviations should be used in the abstract. In addition it is confusing that there are announced 8 different carbon sources which were named GEPS. LEPS and SEPS. Finally, these carbon sources are not clearly displayed in the material and methods part. This must be improved to allow the reader to follow the approach.
what are the functional groups in the EPS? Scleroglucan is a pure bta glucan, without any additional substituents, so only OH groups can be described by IR.
Introduction:
It might be true that the high price restricts the applications of SC, but at the same time it is mentioned that it is widely used in many applications, this is confusing. I think there are also some reviews on scleroglucan available, which can be referenced here.
Check the whole text carefully for mistakes in spacing of references etc. (lane 45, 46, 48 etc.).
lane 41 and following. This part describes results from many different microorganisms and polysaccharides, it is not focused on scleroglucan, what might be much more straight forward. It is a bit confusing to read so many different strains and EPS in that short part, that should be rewritten.
There is also a new manuscript of Farina et al on Scleroglucan production by use of different cheap substrate streams, that might fit here very well.
Lane 54: what is meant by exploring the effects of different carbon sources on the characteristics of EPS and their proteomic differences. Is that referred to the proteom of the cells or scleroglucan. should be rewritten to clarify.
Lane 70 ..was precultured.
Lane 71. mycelia is already the plural, so better ... a large amount of mycelia,
lane 75: Here must be given the 8 different carbon sources tested!
lane 89 - 91: It is not clear how the the samples for rheological measurements were prepared, this must be specified. The sentence: EPS were separated from the fermentation broth ... is confusing here. How can you determine that the EPS was concentrated to 10 g/L?
Lane 98: Not the liquid phase conditions are described if you mention the detector and the column. Please rephrase. Also standards and concentrations mus be mentioned here. In addition a chromatogram would be very helpful to evaluate the results (can be added as appendix).
lne 103: I it really useful to analyse the dried EPS by SEM? AFM analysis of the EPS in suspension would give much better insights in structural differences. SEM just describes the surface of dried smaples, which might depend on various effects, not only structural differences.
lane 108. following: I think there are many references of the original methods available and not only Wangs methods?
lane 114: Not digested, it is more hydrolysed.
lane 117: solume means volume?
lane 123: Check headline : metabolic pathway differences... Rephrase.
lane 123 - 140: The description is not really clear, sequencing can mean also genome sequencing... please rephrase the section to make it clear and straight.
Results:
One very important aspect of EPS is not mentioned in the manuscript. Viscosity mainly depends on the molecular weight of the EPS. So a determination of the molecular weight would be essential to compare them. In general there is nor reason why the basic chemical structure of Scleroglucan should be altered by use of different carbon sources, the only effect might be in a different grade of substitution with 1-6 linked sidechains or the molecular weight. So this aspects must be addressed in the manuscript. I highly recommend to include the molecular weights of the different SC samples.
lane 169: It is not suprising in my opinion that it is only made of glucose. In general the biosynthesis pathways for microbial EPS are highly specific. The change in viscosity will depend much more on the molecular weight.
Table 1 (add chromatogram). In addition check writing of (min). It is fructose and not frucose.
SEM pictures. Was the amount of oxalic acid analysed? Might that affect the different structures. Where the samples treated the same way? LEPS and SEPS seems to be milled, compared to GEPS?
Table 2: It must be explained what - and + means.
lane 194 - 203. It would be possible that oxalic acid coming from glucose binds calcium, and precipitaes with SC. That would explain the high content of calcium in GEPS. In media with fructose the oxalic acid production seems to be minimized, what would explain the low concentration sin SEPS. This should be discussed and taken into account when analysing this data. Schmid et al described the transcriptional effects of various media composition on SC production, so this might be compared with the data of theses experiments.
lane 205 - 224: Only peaks of interets shuld be mention for the IR analysis (uronic acids are not of interest for example). If SEPS is an alpha type, it is not Scleroglucan any more!
lane 226 - 234: I think that many bacterial EPS biosynthesis pathways are very well analysed, there are many references available. I agree that fungal EPS biosynthesis pathways are still a black box, but this should be clearly mentioned and not mixed up here.
lane 238: Why now Lac and Suc group and not SEPS etc? This might be confusing to some readers, so try to introduce the new abbreviation or or be consequent in using the already known ones.
lane 244 - 265: The results should also be linked to oxalic acid production, this might correlate with calcium binding etc.
what was the biomass obtained from the different samples, so it would make sense also to calculate the yield (gEPS/gCarbon source).
I cant see any group (I/II/III) in figure 6, is it figure 5? Than the numbers I/II and II should also be added to the figures, and not a/b and c.
lane 236. What kind of bacteria, when describing scleroglucan production by S. rolfsii?
Also figure 6 is missing any hint to group I/II and III!
In the conclusion nothing is said anymore about that SEPS seems to be an alpha glucan?
