Optimization of Xanthan Gum Production by Demerara Sugar Using Response Surface Methodology

Round 1

Reviewer 1 Report

Sustainability

Article

Optimization of xanthan gum production by demerara sugar using response surface methodology

 

Authors have done this production process to maximize the xanthan production by a complete central factorial composite planning.

Various strains like Xanthomonas axonopodis pv. manihotisIBSBF 290, X. campestris pv. campestrisIBSBF 472 and Xanthomonas sp. S6 from the Cultures Collection of the Biomaterials Laboratory (Institute of Technology and Research, Aracaju, SE, Brazil) were used as xanthan-producing 100 microorganisms.

With adequate figures and tables, the work was presented beautifully.

The literature review was not complete, where several of the recent literature using xanthan were omitted, at least it can be compared with those methods.

Finally evaluated the possibility of supplementing the fermentation medium with mineral salts, using demerara sugar or sucrose as substrates, in XG production. Thereby, sucrose or demerara sugar, K2HPO4, and MgSO4∙7H2O concentration effects on its production were explored by experimental design and response surface analysis. Proposed model studies pointed out a reasonable experimental agreement for xanthan production,  indicating specific nutritional requirements for each Xanthomonas strain. Statistical significance values suggested that K2HPO4 displayed an important role in this production by Xanthomonas 290 and 472 strains, using sucrose or demerara sugar as a carbon source. Regarding the S6 strain, MgSO4∙7H2O displayed a significant role in gum synthesis with sucrose as a carbon source.

Accept with minor mandatory changes.

Author Response

Many thanks for the suggestions! Changes are in “Track Changes” throughout the manuscript.

Added some recent work using Xanthan in the introduction of the work, please check the Manuscript.

 

“In addition, the adjustment of the strains with the conditions applied in the fermentation are important has a direct influence on the characteristics, techniques, yields, compositions and structures of XG [10,18] of the XG produced, since the strains present different productivity depending on the factors implemented during the fermentation [19], as it has a direct influence on the characteristics, techniques, yields, compositions and structures of XG. The Xanthomonas strains most used in the literature to produce XG are X. campestris pv. campestris 629, X. campestris pv. campestris 1078, X. campestris pv. campestris S6, X. campestris pv. campestris 254, X. sp. 1537, X. campestris pv. campestris 729, X. campestris pv. campestris 607, X. campestris pv. campestris 1167, X. campestris pv. mangiferaeindicae 1230, X. campestris pv. arracia 1198, X. axonopodis pv. manihotis 1182, X. melonis 68 [20,21].”

 

“Screening research on alternative or conventional fermentation media for this polymer synthesis has been reported [7,34] for example agricultural residues and biomass with lignocellulosic content [22] (such as tapioca pulp [35], sugarcane bagasse [36], orange peel [37], kitchen waste [38], rice bran [39], chicken feathers [40], coconut shell, cocoa husk [41], potato crop [42], cellar wastewater [43] and corncob [21], however, these works found some limitations in pre-treatments and production yields. Furthermore, no reports describe the use of demerara sugar in XG production. This raw sugar appears to be a promising source since its chemical composition displays adequate nutritional requirements for a fermentative medium.”

Reviewer 2 Report

General comments

The current study represents the statistical optimization of the microbial production of xanthan sugar from three Xanthomas species using sucrose or demerara sugar. The study is well-written in a scientific way. However, I reject the current study for the following reasons:

1-      The rationale and novelty behind the study are not clear.

2-      The study represents a lot of repeated and similar results (three strains, 2 designs for each strain) without any obvious impacts on the study outcomes. All these experiments could be conducted in one experiment using one variable at a time (OVAT) to decide which the potent organism (among three) and which is the optimum sugar (sucrose or demerara) then you conduct one statistical design to detect the real optimum conditions

3-      The study is also based on the application of two edible sugars (sucrose and demerara), which is totally against sustainable production.    

4-      The study lacks any analysis of the produced polysaccharide. How can you be sure that the fermentation product is xanthan gum?

5-      No application was represented for produced polysaccharides.

Minor comments

-     Line 108; Space between (250 mLErlenmeyer)

-     In table 1; three repeated ranges were represented for the three organisms. As the values are identical (not giving any additional information) it should present one time for the three organisms

Author Response

Many thanks for the suggestions! Changes are in “Track Changes” throughout the manuscript.

• The rationale and novelty behind the study are not clear. 

The authors thank you for your collaboration and suggestions. As for the logic and novelty of the work, the topic addressed refers to the alternative use of low-refined sugar to produce XG and its influence on yield. Furthermore, it is noteworthy that the parameters were studied for three different strains of Xanthomonas.

2-      The study represents a lot of repeated and similar results (three strains, 2 designs for each strain) without any obvious impacts on the study outcomes. All these experiments could be conducted in one experiment using one variable at a time (OVAT) to decide which the potent organism (among three) and which is the optimum sugar (sucrose or demerara) then you conduct one statistical design to detect the real optimum conditions

• The study is also based on the application of two edible sugars (sucrose and demerara), which is totally against sustainable production.    

Taking into account that Brazil is a sugar cane producing country, it has an abundance of the product, and, in addition, demerara sugar does not have a low level of refinement, and this proved to have a positive effect on the production of XG, as the salts present improve fermentation and have great effect on the production of XG.

• The study lacks any analysis of the produced polysaccharide. How can you be sure that the fermentation product is xanthan gum? 

This study is not aimed at characterizing the polysaccharide, but at optimizing production. As for the certainty that the product synthesized in this work is GX, it is enough to know that the only product synthesized by the bacteria studied in this work is XG.

• No application was represented for produced polysaccharides. 

Thank you for your comments and I would like to explain that the purpose of this work was to optimize the production of XG demerara sugar as a carbon source, as well as to evaluate the best strain of Xanthomonas. The application will be explored in future work.

Minor comments

-     Line 108; Space between (250 mLErlenmeyer)

Space has been added.

-     In table 1; three repeated ranges were represented for the three organisms. As the values are identical (not giving any additional information) it should present one time for the three organisms 

The table was adjusted, and repeated data were removed.

Reviewer 3 Report

Manuscript is good but needs some clarification on my given comment.

Comments for author File: Comments.pdf

Author Response

Many thanks for the suggestions! Changes are in “Track Changes” throughout the manuscript.

1. Abstract is good but more findings can be included.

The authors appreciate the suggestions! More results have now been added to the summary. please check in the manuscript.

“The strains of Xanthomonas 472 and S6, using demerara sugar and higher concentrations of salts, exhibited a higher yield of XG (36 and 32%) than when using sucrose and the same concentration of salts.”

2. In Introduction the line “Although screening research on alternative or conventional fermentation media for this polymer synthesis has been reported [3,25] no reports describe the use of demerara sugar in xanthan production” The Author though this statement want to state that this work is dine first time?

Yes, this work is new, there is nothing described in the literature for demerara sugar and XG production. This sentence has been rewritten for better understanding. Please check in the manuscript.

“Screening research on alternative or conventional fermentation media for this polymer synthesis has been reported [3,30] for example agricultural residues and biomass with lignocellulosic content [18] (such as tapioca pulp [31], sugarcane bagasse [32], orange peel [33], kitchen waste [34], rice bran [35], chicken feathers [36], coconut shell, cocoa husk [37], potato crop [38], cellar wastewater [39] and corncob [17]), however, these works found some limitations in pre-treatments and production yields. Furthermore, no reports describe the use of demerara sugar in XG production.”

3. Introduction is written nicely but lack the proper justification of present work, where is methodology adopted with reference to other studied or not

The Introduction has been improved and the justifications for this work have also been added. Please check in the manuscript.

4. In Section 2.3. “After fermentation to evaluate the xanthan production, cells were removed from the 117 fermented broth by centrifugation (9626 × g/5°C/60 min). The biopolymer was recovered 118 from cell-free supernatant by precipitation with 98% ethanol in a ratio of 3:1 (v/v). The 119 precipitated gum was collected and dried at 30 ± 2°C for 72 h. Its production was ex- 120 pressed as g∙L-1 (grams of XG per liter of fermentation broth)”Author should elaborate the processs.

This sentence has already been rewritten for better understanding.

5. Line in Introduction section “Bacteria were grown on MY-agar medium with the following composi- 101 tion (g∙L-1 ): yeast extract, 3.0; malt extract, 3.0; peptone, 5.0; glucose, 10.0 and agar, 20.0, 102 and subcultured every three weeks. After incubation (25°C/48 h), cultures were stored at 103 4°C Inoculum cultures were prepared in MY liquid medium in 125 mL Erlenmeyer 104 flask containing 14 mL of culture medium at 28°C, 100 rpm for 24 h until reaching a log 105 growth of 1011 CFU/mL.” needs reference add the below reference

Influence of particle size on physical, mechanical, thermal, and morphological properties of

https://doi.org/10.1007/s00289-022-04214-1

The reference has already been added.

6. Section 2.4. Experimental design and data analysis. For the data analysis component are not clear/missing in the text. Author should clarify it in the material method section.

Section 2.4 has been rewritten for clarity.

7. Authors have given better figures for understands of viewers in various place but in few place it is bit not clear.

The figures have been adjusted

8. Section 3 and 4 are well explained and results are clear to understand but section 2 should be rewritten

Section 2 has been rewritten for better understanding.

9. As per the results findings the Conclusion given is short, it should be a complete gist of study in bullet points.

Conclusions have been improved.

Reviewer 4 Report

The aim of the work was to optimize xanthan gum production using demerara sugar by means of RSM. The introduction is quite simple, but leads well to the topic and justifies the undertaken research. The experiential part is “packed” with statistics, but lacks scientific discussion of the results. This is the weakest part of the  manuscript, the authors focus on the RSM, but do not discuss the results and their implications. Conclusison cound be more detailed, with data regarding optimal conditions.

Author Response

Many thanks for the suggestions! Changes are in “Track Changes” throughout the manuscript.

Discussions were added with the results obtained in the study to improve the results and discussions as requested.

“The concentration of demerara or sucrose sugar, the concentration of MgSO₄∙7H₂O, and K₂HPO₄ are important factors that influence the efficiency of the fermentation process for XG production. The conditions of these experiments and the results obtained are listed in Table 2.

As seen in Table 1, the lowest XG production was achieved with the strains Xanthomonas S6 in experiment 2 (0.0600 g∙L^-1) where lower concentrations of demerara sugar and MgSO₄∙7H₂O were used, and higher concentrations of K₂HPO₄. Silva et al. [46], also did not find significant effects in relation to the concentration of MgSO₄∙7H₂O, in addition, the presence of K₂HPO₄ improved the yield of XG production. The improvement in extraction was observed when the MgSO₄∙7H₂O concentration increased up to 1 g∙L^-1 in experiment 3, where the highest XG production in this study was obtained for strains 472 and S6 (1.3075 and 1.3839 g∙L^-1, respectively). These results agree with Jesus et al. [21] which achieved higher yields of XG production using the S6 strain supplemented with salts and hemicellulose fraction obtained by alkaline hydrolysis of corncob. Furthermore, MgSO₄∙7H₂O at lower concentrations 0.2 g∙L^-1and 30 g∙L^-1 of demerara sugar, showed similar results for strain 472. It was verified that the highest productions of XG for strains Xanthomonas 290 and 472, when using sucrose as the main source of carbon, were obtained in tests containing higher levels of K₂HPO₄ and sucrose (experiment 6, 0.6840 and 1.0034 g∙L^-1, respectively). However, for strain 290 the best results were obtained in the conditions applied in the central points (50, 0.6 and 0.50 gL^-1 of demerara sugar, MgSO₄∙7H₂O and K₂HPO₄, respectively. In addition, the lowest concentration of XG produced by this strain using demerara sugar was obtained in experiment 8 or more, there were no significant differences be-tween the other experiments. Other authors [47] state in the literature that the most promising culture medium to produce XG is sucrose supplemented with salts. On the other hand, in this study it is observed that demerara sugar has a higher XG production compared to experiments with the same conditions using sucrose as a carbon source, making it a very important factor to produce XG.”

The conclusions of the work were also improved.

“The three selected strains displayed different influence from the fermented conditions for the xanthan production. Statistical significance values suggested that sugar concentration and K₂HPO₄ displayed an important role for this production by Xanthomonas 472 strain, using sucrose or demerara sugar as a carbon source. Regarding the S6 strain, MgSO₄∙7H₂O displayed a significant role in gum synthesis with sucrose but not with demerara as a carbon source. With strain 290 the demerara sugar concentration increases XG but with sucrose as carbon source the production is higher with K₂HPO₄. The evaluated salts are important factors for XG production, and the demerara sugar can partially replace this mineral salt requirement. Economically, experimental data show the

Many thanks for the suggestions! Changes are in “Track Changes” throughout the manuscript.

relevance of demerara sugar as an efficient alternative carbon source in xanthan production.”

 

Round 2

Reviewer 4 Report

The manuscript has been improved and can be reccoemended for publication.