Fermentative Hydrogen Production from Lignocellulose by Mesophilic Clostridium populeti FZ10 Newly Isolated from Microcrystalline Cellulose-Acclimated Compost
, Baoxuan Jiang 1,2, Haokun Zhang 1,2, Sitong Qian 1,2, Tao Wei 1,2,*, Zhiping Zhang 1,2, Lili Song 1,2 and Xu Yang 1,2
Round 1
Reviewer 1 Report
The Manuscript entitled: “Fermentative hydrogen production from lignocellulose by mesophilic Clostridium populeti FZ10 newly isolated from microcrystalline cellulose acclimated compost” (ID applsci-1919211) requires revision before accepted for publication. The specific comments are given below.
1. Abstracts needs to have more precision as in the current form it appears. In the abstract, please add an indication of the achievements from your study that is relevant to the journal scope.
2. Provide significant words which are more relevant to the work in a logical sequence as ‘keywords’.
3. The "Introduction" section should follow the state of the art of this field and review what has been done, for supporting the research gap and the significance of this study. Please improve the state of the art overview, to clearly show the progress beyond the state of the art.
4. In the last paragraph of the introduction, clearly indicate the research hypothesis.
5. Ln 90: Why was this medium composition used? Does it result from the literature data?
6. Ln 122: What were the columns and detector? What temperatures were the sample injection and detector ports?
7. Include standard deviations in all your results.
8. Statistical analyzes are very important in research manuscripts. How were the normality of the distribution and the differences between the variables found?
9. Improve the quality of figures.
1No clear conclusions from this study. More details should be included in the conclusion.
1It is also recommended to discuss and explain what should be the appropriate policies based on the findings of this study.
Comments for author File: 
Comments.pdf
Author Response
Response to Reviewer 1 Comments
Point 1: Abstracts needs to have more precision as in the current form it appears. In the abstract, please add an indication of the achievements from your study that is relevant to the journal scope.
Response 1: According to reviewer’s suggestion, the indication of the achievements from our study that is relevant to the journal scope has been added to section Abstract :”The discovery of strain C. populeti FZ10 has special significance in the field of bioenergy.”
Point 2: Provide significant words which are more relevant to the work in a logical sequence as ‘keywords’.
Response 2: According to reviewer’s suggestion, the “Keywords” has been revised :”Clostridium populeti FZ10; Isolation and identification; Dark fermentation; Hydrogen production; Lignocellulose”
Point 3: The "Introduction" section should follow the state of the art of this field and review what has been done, for supporting the research gap and the significance of this study. Please improve the state of the art overview, to clearly show the progress beyond the state of the art.
Response 3: According to reviewer’s suggestion, we review the research work which have been done in this field and add it to the section Introduction (page 2 Line 61-66) :”Lo et al. isolated a mesophilic Clostridium which could effectively utilize sucrose and xylose to produce hydrogen, the hydrogen yield reached 129.1 mL/g sucrose and 172.9 mL/g xylose, respectively. But the strain could not directly utilize cellulose to produce hydrogen [24]. Magnusson et al. found a thermophilic Clostridium also faced with the same problem of low hydrogen yield from cellulosic biomass. The maximum hydrogen yield of this strain from grain was only 23.97 mL/g [25].”
Point 4: In the last paragraph of the introduction, clearly indicate the research hypothesis.
Response 4: According to reviewer’s suggestion, the research hypothesis has been supplemented in the last paragraph of the Introduction (page 2, Line 73-75) :”Hydrogen production Clostridium could efficiently convert cellulosic substrate into hydrogen, which could be isolated through microbial acclimation, the limitation as stated above would be overcome.”
Point 5: Ln 90: Why was this medium composition used? Does it result from the literature data?
Response 5: The composition of the medium used in the experiments was based on the results of our previous studies. We also referenced some literatures. [Pan, C.M. et al. Int. J. Hydrogen Energy 2011, 36, 4852-4862.] and [Song, Z.X. et al. Bioresour. Technol. 2014, 157, 91-97.]
Point 6: Ln 122: What were the columns and detector? What temperatures were the sample injection and detector ports?
Response 6: The detailed information of Gas Chromatography have been supplemented in section 2.5. (page 4, Line 138-146) :”Hydrogen content was measured by a Gas Chromatograph (GC, Agilent 7890B) equipped with a thermal conductivity detector (TCD) and a porapak Q stainless column. The temperatures of the injection port, the oven and the detector were 100℃, 80℃ and 150℃, respectively. Nitrogen served as the carrier gas at a flow rate of 20 mL/min. The concentrations of volatile fatty acids and alcohols were detected using another GC with a flame ionization detector (FID) and an 8-ft stainless column packed with 10% PEG-20 M and 2% H3PO4 (80/100 mesh). The temperatures of the injection port, FID detector, and oven were 220℃, 140℃, and a programmed column temperature of 115-170℃, respectively. Nitrogen was used as the carrier gas at a flow rate of 20 mL/min [34].”
Point 7: Include standard deviations in all your results.
Response 7: According to reviewer’s suggestion, standard deviations have been added to the revised manuscript.
Point 8: Statistical analyzes are very important in research manuscripts. How were the normality of the distribution and the differences between the variables found?
Response 8: One-way ANOVA analysis was employed to analyze the signification of the difference (P-values < 0.05), and all the experiments were carried out at least three parallel experiments.
Point 9: Improve the quality of figures.
Response 9: According to reviewer’s suggestion, we have enlarged the fonts of all Figures and used larger Figures in the revised manuscript.
Point 10: No clear conclusions from this study. More details should be included in the conclusion.
Response 10: According to reviewer’s suggestion, some experimental details have been added to the section Conclusion :”a mesophilic anaerobic hydrogen-producing Clostridium had been successful isolated from cow dung compost acclimated using microcrystalline cellulose in an anaerobic bioreactor,” and “such as endoglucanase, exoglucanase, β-glucosidase and xylanase”.
Point 11: It is also recommended to discuss and explain what should be the appropriate policies based on the findings of this study.
Response 11: According to reviewer’s suggestion, we added some discussion on strategies to improve the fermentative hydrogen production from lignocellulose in section Result and discession (page 11, Line 341-345) :”Obtaining high efficiency hydrogen-producing strains is the key to improving the efficiency of hydrogen production. In this study, the methods of microbial pretreatment and microbial acclimation were used for the screening of hydrogen-producing bacteria. These are effective strategies to obtain hydrogen-producing strains that efficiently convert lignocellulosic biomass into hydrogen.”
Author Response File: 
Author Response.docx
Reviewer 2 Report
In this work, Fermentative hydrogen production from lignocellulose by mesophilic Clostridium populeti FZ10 newly isolated from microcrystalline cellulose acclimated compost is presented. The manuscript is well prepared, however, few queries should be addressed, before considering it for publication:
1) Line 42-42: Please provide references for mentioned chemical, physical and biological methods.
2) Line 47-49: Authors mention enzymes, responsible for hydrolysis, please name some of the most important enzymes, that are involved in such process and provide references.
3) Line 90: it would be better to present the composition of the basal medium in a table, please consider
4) Line 111-14: consider putting the fermentation medium into table again for more clear presentation
5) Line 116: delete excess space between “(CS),” and “wheat”
6) Line 125: How was SEM performed, were the samples prepared with Au? More detailed description needed.
7) Line 128: Add description of filter paper assay method and how is the cellulase activity by this method evaluated?
8) Figure 2 and 3: Please consider making the figure bigger and more clearly to read
9) Figure 4 caption: use capital letter for “Cumulative”
10) Please consider bigger font for all figures
11) Figure 5 caption: use capital letter for “Cumulative”
12) Line 240: utilize
13) Line 251: no need to write out SEM, use only abbreviation SEM
14) Line 255: the waxy layer “was” almost removed?
15) Figure 6: use only abbreviation SEM and capital letter for unfermented
16) Link 274: obviously?
17) Figure 7: use capital letter for Chemical
18) Line 300-311: the space between the lines is different (bigger), please correct. Also, in the following table 3 and in Conclusions.
19) Line 319: please add, which hydrolytic enzymes can be secreted by your strain.
Considering these minor changes to the manuscript, I think the overall research work was nicely and adequately performed and can be considered for publication in journal Applied Sciences.
Author Response
Response to Reviewer 2 Comments
Point 1: Line 42-42: Please provide references for mentioned chemical, physical and biological methods.
Response 1: According to reviewer’s suggestion, the references 8-11 for pretreatment methods have been supplemented to the Introduction section.
Point 2: Line 47-49: Authors mention enzymes, responsible for hydrolysis, please name some of the most important enzymes, that are involved in such process and provide references.
Response 2: According to reviewer’s suggestion, some important enzymes and related references 12-14 have been added to the section Introduction (page 2, Line 49-51) :”such as endoglucanase, exoglucanase, cellobiohydrolase, xylanase, β-xylosidases, etc.”
Point 3: Line 90: it would be better to present the composition of the basal medium in a table, please consider
Line 111-14: consider putting the fermentation medium into table again for more clear presentation
Response 3: Thanks a lot for reviewer’s suggestion. According to suggestion, the chemical composition of the basal medium, the basal fermentation medium and the nutrient solution are listed in Table 1, Table 2, and Table 3, respectively.
Point 4: Line 116: delete excess space between “(CS),” and “wheat”
Response 4: According to reviewer’s suggestion, the excess space between “(CS)” and “wheat” has been deleted.
Point 5: Line 125: How was SEM performed, were the samples prepared with Au? More detailed description needed.
Response 5: According to reviewer’s suggestion, the detailed operation procedure of SEM has been added to section 2.5. (page 4, Line 149-151) :”All samples were dried at 60℃ to a constant weight, and samples were affixed using conductive tape. In order to enhance the electric conduction of samples, the samples were sputtered with a layer of gold.”
Point 6: Line 128: Add description of filter paper assay method and how is the cellulase activity by this method evaluated?
Response 6: According to reviewer’s suggestion, the detailed description of filter paper assay method has been added to section 2.5. (page 4, Line 154-158) :”The assay system had a total volume of 2 mL, consisting of 1 mL of culture supernatant, 1 mL citrate acid buffer (0.05 M, pH 5.0) and 50 mg of Whatman filter paper No. 1, incubated for 60 min at 50℃. The unit of cellulase activity was defined as the amount of enzyme that produced micromoles of reducing sugar per min.”
Point 7: Figure 2 and 3: Please consider making the figure bigger and more clearly to read
Response 7: According to reviewer’s suggestion, Figure 2 and 3 have been enlarged.
Point 8: Figure 4 caption: use capital letter for “Cumulative”
Figure 5 caption: use capital letter for “Cumulative”
Figure 7: use capital letter for Chemical
Response 8: According to reviewer’s suggestion, the captions of Figure 4, Figure 5 and Figure 7 have been revised.
Point 9: Please consider bigger font for all figures
Response 9: According to reviewer’s suggestion, the fonts in all figures have been enlarged.
Point 10: Line 240: utilize
Response 10: According to reviewer’s suggestion, the word “utilized“ has been replaced with “utilize“.
Point 11: Line 251: no need to write out SEM, use only abbreviation SEM
Response 11: According to reviewer’s suggestion, the phrase “scanning electron microscopy (SEM)“ has been replaced with “SEM“.
Point 12: Line 255: the waxy layer “was” almost removed?
Response 12: According to reviewer’s suggestion, the sentence “The waxy layer on the surface almost removed“ is changed into “The waxy layer on the surface was almost removed“.
Point 13: Figure 6: use only abbreviation SEM and capital letter for unfermented
Response 13: According to reviewer’s suggestion, the caption of Figure 6 has been revised.
Point 14: Link 274: obviously?
Response 14: According to reviewer’s suggestion, the word “obvious” is changed into “obviously”.
Point 15: Line 300-311: the space between the lines is different (bigger), please correct. Also, in the following table 3 and in Conclusions.
Response 15: According to reviewer’s suggestion, inappropriate space between the lines has been adjusted in the revised manuscript
Point 16: Line 319: please add, which hydrolytic enzymes can be secreted by your strain.
Response16: According to reviewer’s suggestion, the hydrolytic enzymes secreted by strain FZ10 have been added to section Conclusion (page 11, Line 352-353) :”(such as endoglucanase, exoglucanase, β-glucosidase and xylanase)”
Author Response File: Author Response.docx
Reviewer 3 Report
The manuscript applsci-1919211 is worth considering. The title is accurate and informative. The article is generally well-written and the discussion offers extensive comparison with the literature on the topic. As carbon sources for FZ10, the authors use i) certain carbohydrate polymers, and ii) biomass, more suitable in terms of circular bioeconomy. The results of hydrogen production from this particular strain are promising and deserve publication after some formal modifications, mainly in what pertains to the figures and tolerance/confidence intervals.
Please find the PDF file attached! Besides text, it has some images that were important to convey my message.
Comments for author File: Comments.pdf
Author Response
Please see the attachment.
Author Response File: Author Response.docx
Round 2
Reviewer 1 Report
Thank you for considering my suggestions.
