Synergistic Enhancement Effect of Compound Additive of Organic Alcohols and Biosurfactant on Enzymatic Hydrolysis of Lignocellulose

Reviewer1: 

Dear reviewer, thanks for your careful checks. We are sorry for our carelessness. Based on your comments, we have made the corrections to make the word harmonized within the whole manuscript.

Question

Response

1.  English language need to be improved (Example from the abstract: ”... The enzymatic hydrolysis efficiency was relatively low due to the insufficient of cellulose degradation enzyme components, especially for woody biomass. In the present study, organic alcohols (OA) and biosurfactant could synergism impact on...”

ü We apologize for the language problems in the original manuscript. This manuscript have checked by a native English-speaker.

ü We are agree with the comment and reformulated the phrase in the revised manuscript. 

2. Page 2, rows 46-48. The formulation of phrase indicate that authors does not correctly present the enzymatic complex referring (...”However, this enzyme (BLG) is deficient in cellulase components”...)

ü The description of BGL and it’s key role on enzymatic hydrolysis was added.

3. Row 75: XYL abbreviation was not defined

ü Abbreviation of xylanase was defined as XYL.

4. Page 2, rows 96-98, carbon source not mentioned

ü Description of carbon source has been added.  And more details in preparation procedures for W₆₃ fermentation on BGL and XYL production are described in our previous report (Liang et al., 2020).https://doi.org/10.1016/j.biortech.2020.124019

5. Rows 105-106: ”...acid-treated poplar was washed with distilled water to pH neutral, and oven-dried...”. Other researches indicate that by drying the pretreated lignocellulose, the pores obtained by pretreatment in lignin will collapse, reducing the access of cellulolytic enzymes to cellulose – hence, the pretreated biomass should not be dried before hydrolysis. This is an important aspect that need to be discussed briefly in this section.

ü We are gratefully thanks for reviewer’s expert advice that by drying the pretreated lignocellulose, the pores obtained by pretreatment in lignin will collapse, reducing the access of cellulolytic enzymes to cellulose. Now we deeply realize that drying the pretreated lignocellulose will seriously affect the conversion rate of cellulase enzymatic hydrolysis. However, this study was focused on different compound additives on the enzymatic hydrolysis effects of lignocellulose. When pretreated substrate drying at low temperature  can reduce the hornification effect. And the dry substrate is easy to storage and does not easy to grow mildew and kept stale. Hence, we used the dry pretreated poplar for enzymatic hydrolysis in this study.

6. Page 9, row 299-300: please reformulate: ”... BGL activity and stability were difference...”

ü We are agree with the comment and rewrote this sentence.

7. Well-described results showed that the association of some additives improve the degradation of Avicel and especially pretreated poplar and stabilize the two kinds of enzymes tested here in the hydrolysis process. However, an economic analysis regarding the overall process (cost of additives and value resulted by increased sugars yields) would be more than welcome in the future.

ü We sincerely thank the reviewer’s valuable feedback we have used to improve the quality of our manuscript. However, experiments we are operated were in small scales. Further studies will carry out to clarify the mechanisms and the economic analysis for lignocellulose hydrolysis process in our future work.

Reviewer2: The article fermentation-2007244 presents serious errors in the use of the English language. I have hardly found a sentence that could be understood in the abstract and introduction section. Furthermore, the use of the two substrates, much less the auxiliary enzymes in the introduction section was not justified. This work must be rejected as it is incomprehensible and without merit to be published in this prestigious journal.

Response:

Dear reviewer, we sincerely invite reviewer to comment again on our revised manuscript.

1. We apologize for the language problems in the original manuscript. The language presentation was improved with assistance from a native English speaker with appropriate research background.

2. We agree with the comment and rewrote the abstract and introduction as well as the whole manuscript.

3. This study focus on different compound additives on the enzymatic hydrolysis effects of lignocellulosic biomass. The selected crystalline cellulose of Avicel and woody biomass of poplar were more compactable and hard to decomposition. Waste poplar, a woody manufacturing residue, can be used as a suitable biorefinery feedstock because poplar is a fast growing and widely distributed woody species in the world. It has a matrix composed of three major biopolymeric components: lignin, hemicelluloses and cellulose. Thus, we are aiming to find an excellent compound additives, synergic with cellulase and auxiliary enzyme cocktail on the improvement of enzymatic hydrolysis for lignocellulose conversion of biofuels.

Reviewer3: The papers proposes the improvement of enzyme hydrolysis with the use of different additives. The work is extense and great results are obtained. However, sometimes it is difficult to follow, considering there are two enzymes cocktails, you evaluate several enzymes and you use several additives. I would highly recommend to improve the material and method section but, mainly, I would make an important effort to explain better the results. The discusion of Figure 4 is difficult to follow. The information related to this figure included in the abstract and conclusion sections is also not very clear. 

Response

Dear reviewer, thanks for your careful checks. We are sorry for our carelessness. Based on your comments, we have made the corrections to make the word harmonized within the whole manuscript. We had improved the material and method section as well as abstract and conclusion sections in details. Moreover, we had redrew Figure 4 on the enzyme relative decreasing/increasing activity for more clearly discussion.

Question

Response

1. Line 16-18. Revise this information

In the association of N-decanol (C10) and sophorolipid (SL) increased the pretreated poplar enzymatic conversion rate from 17.9% to 85%, improved by 67.1%. Moreover, it was found that the mechanism of compound additives stimulated the enzyme activities and stabilize the enzyme performance for commercial cellulase and Hypocrea sp. W63 beta-glucosidase (BGL) and xylanase (XYL) rich cellulase cocktail.

ü We are agree with the comment and abstract has been revision.

2. Line 39-40. You could include normal composition of Woody biomass (cellulose, hemicellulose and lignin). Also, it could be interesting to include the source of this biomass.

ü We sincerely thank the reviewer for careful reading. Normal composition of woody biomass has been added.

ü The source of this woody biomass has been introduced.

3. Line 48. What do you mean with “However, this enzyme is deficient in cellulase components”

ü BGL is a key enzyme in the sugar-enzyme platform from lignocellulose degradation. Hence, it is involved in the important biological degradation of cellobiose and other soluble oligosaccharides into glucose. However, most cellulases that are used for the commercial lignocellulosic hydrolysis, are produced by filamentous fungi such as the main producer Trichoderma sp., which are short for BGL

4. Line 50. Include normal problema associated with solvent hydrolysis

ü Challenge of enzymatic hydrolysis process has been introduced.

5. Line 86-88. The phrase needs revision

ü We are agree with the comment and this phrase has been revision.

6. Line 89. Is cellulase LLC02 a purified cellulase or is an enzyme mixture?

ü LLC02 cellulase is an commercial cellulase mixture.  

7. Line 93. How was stored?, frozen, lyophilized?

ü BGL was stored at 4 ^oC then used for the following experiments.

8. Line 94. How was inoculated?

ü The fungal strain was inoculated into a 150 mL Erlenmeyer flasks with 50 mL of potato-dextrose liquid medium, as seedling for the fermentative production of BGL and XYL at 30 °C, 150 rpm for 3 days. After that, 10%(v/v) seed culture of W₆₃ was inoculated into a 250 mL Erlenmeyer flasks with 100 mL of fermentation medium.

9. Line 95. Give more information of the inoculum preparation for the subsequent fermentation and the % of inoculum added in the fermentation. Also it should be included the type of material used for the inoculum and fermentation (flasks, fermenters,…)

ü We feel sorry for our carelessness. In our resubmitted manuscript, more details about the inoculum preparation and fermentation were supplemented.

10. Line 99. I would use another name different to BGL solution as I supponse several enzymes were produced.

ü We sincerely thank the reviewer for careful reading. “BGL solution” was take “W₆₃ crude enzyme solution” alternative

11. The meaning of FPU and CBU should be included in the manuscript

ü The meaning of FPU has been supplemented in materials section by  commercial mixture cellulase LLC02 Filter Paper Activity which appear for the first time.

ü We are agree with the comment. W₆₃ crude enzyme solution rich for BGL and XYL. Here it is revised to the enzyme protein concentration.

12. Line 116. Include the meaning of I5 and P8

ü We sincerely thank the reviewer for careful reading. As suggested by the reviewer, OA of isoamyl alcohol and phenyl ethanol were marked as I5 and P8 in the materials section.

13. Line 117. Taps?

ü We feel sorry for our carelessness. An explaination was that the experiment without any OA was used as a control.

14. Line 122. Change V/V

ü We sincerely thank the reviewer for careful reading. As suggested by the reviewer, “V/V” has been revised to “v/v” throughout this manuscript.

15. Line 122. Why did you only study C1, C4, C5, I5 or C10?

ü Because of C1, C4, C5, I5 and C10 have the greatest promotion effect on enzymatic hydrolysis screening with Avicel as substrate.

16. Line 125. Phrase needs revision

ü We are agree with the comment and this phrase has been revision.

17. Line 125. Why did you use 0.1 g of poplar and no 1 g as you did with Avicel?. Why did you use different enzymatic units in the hydrolysis of poplar?. Also, why did you use different working volumen?

ü Because pretreatment of raw poplar material is not easy to prepare, and this experiment was to investigate the effects of different compound additives on the enzymatic of different concentrations of different substrates.

ü Experiment was used W₆₃ crude enzyme solution which is rich for BGL and XYL. Here it is revised to the enzyme protein concentration.

18. Line 133. Did you do this dilution for the experiment or for the enzyme activity determination?

ü Yes, we have did a preliminary experiment for the dilution of LLC02 and W₆₃ for FPase, CMCase, pNPGase and Xylanases activity.

19. Line 137. Why did you measure xylanase activity?

ü Because W₆₃ crude enzyme is rich for BGL and XYL activity.

20. Section 2.3.5. Explain which activities you measure in the commercial extract and in the enzyme extract you produced.

ü We have measured all the activities including FPase, CMCase, pNPGase and Xylanases for LLC02 and W₆₃, respectively.  

21. Line 142. What is XYL rich cellulose?

Were hydrolysis performed in duplicate?

Line 142. Which volumen of the enzyme mixture and wich volumen of additive was mixed with the buffer?. This information is not clear.

ü We are agree with the comment. This phrase was revision.

ü Each sample was evaluated in triplicate, and the results was showed in Figure 4 with error bar.

ü The information of different combination additives have been added.

22. Line 216. The fact you include “addition of W63 BGL” is confusing as all the hydrolysis are carried out with W63 BGL

ü W₆₃ crude enzyme solution which is rich for BGL and XYL. Here it is revised to the enzyme protein concentration.

23. Line 217. Compared is written in capital letter

ü We sincerely thank the reviewer for careful reading. This sentence contains a punctuation error and has been revised.

24. Line 222-223. You mentioned that there is not difference between T3 and T4. However, you mentioned that the enzymatic hydrolysis conversion was higher in T4 than in T3.

ü We are agree with the comment. This phrase has been revised.

ü There was not much difference in the cellulose conversion rate at 72 h for the OA isomers between T3 and T4. The cellulose conversion rate of T3 and T4 were 34.29% and 34.71%, respectively.

25. Line 226. The order you stablish was done considering the data at 72 hours. This should be mentioned.

ü We are agree with the comment and this phrase has been revised.

26. Line 227. What do you mean with “this association”?

ü This phrase has been revised. The word “association” was change to “combination”.

27. Line 227-230. This information is not completely clear.

ü We are agree with the comment and this sentence has been revised.

28. Line 239. Why did you use different FPU/g with Avicel and with poplar?. You compare the results of both substrates in line 242-243 but they hydrolysis have not been carried out in the same conditions.

ü Avicel and poplar enzymatic hydrolysis used the same filter paper enzyme activity of 10 FPU/g substrate. 

ü This part is for mechanism study. The effect of enzyme activity and stability under the condition of different additives requires standard detection under the condition of a certain concentration dilution, so as to compare the relative enzyme activity and the changed of enzyme stability. However, the standard detection of different enzyme activity uses different substrates and different concentrations. In addition, the FPA activity used in the enzymatic hydrolysis experiment of Avicel and poplar was only 10 FPU/g substrate. With the consumption of enzymatic hydrolysis, it was difficult to detect various enzyme activities and their stability, so the same amount of cellulase can not used in the same conditions.

29. Line 252. Explain in which conditions you obtained this order as in the graph, at 72 h, the yields of T2 are higher than those of T1. Revise this order.

ü In figure 4a, the colour light blue represent FPA activity whereas the colour dark blue represent FPA stability. It is cleared that T1’s relative FPA activity and stability were superior than T2.

ü The cellulase enzymatic hydrolysis is a complicated process, and the filter paper activity of cellulase cannot represent the enzymatic hydrolysis effect on lignocellulosic biomass. Different substrates and different compound additives exist a certain relationship with the cellulase enzymatic hydrolysis process as well as the collaborative reaction process of auxiliary enzyme. Our current research results are just a preliminary conclusion of this study, and of course we will do further study in the future.

30. Line 252-253. Phrase needs revision

ü We are agree with the comment and this phrase has been revised.

31. Line 253. Why these differences with isomers are more important in poplar than in Avicel?

ü That’s because poplar is a kind of lignocellulose has a complex structure with numbers of pores. Whereas Avicel is one of a pure cellulose has a highly crystalline structure. Isomers of organic alcohols combinated with sophorolipid could form different hydrophobic environment lead to affects the enzymatic hydrolysis process. Hence it is important to evaluate the difference between poplar and Avicel enzymatic hydrolysis in presence of different compound additives.

32. Line 256-257. This information is not clear.

ü We are agree with the comment and this phrase has been revised.

33. Line 277. You mentione “Moreover, W63 FPA stability of all the samples did not  decrease due to the presence of compound additives”. I do not understand what you are comparing to make this statement. If you compare the relative activity of the activity and the stability (light blue vs dark blue), there is a decrease in all the cases, which I think it should be explained with the loss of activities. You should explain better in material and methods the difference of the metodology used to get the results of activity and stability to evaluate the differences. Also the information included in lines 277-278 should be clarified.

ü The sentence about W₆₃ FPA stability was revised.

ü The loss of stability on W63 has been explained.

ü The difference of the metodology used to get the results of activity and stability to evaluate the differences were further explained.

ü The information of BGL activity and stability on LLC02 and W63 have been supplement.

34. Line 278-280. Phrase needs revision.

ü We are agree with the comment. This phrase has been revision carefully.

35. Line 284-286. Phrase needs revision. Explain also what happened with enzyme stability as there is not difference.

ü This phrase has been revision and the explanation of XYL stability by different compound additives were gave.

36. Line 290-292. Elaborate more this information.

ü More information about XYL activity and stability of W₆₃ was supplement.

37. Line 293. You mentione “….protect the endoglucanase activity of LLC02 and stabilize the enzyme activity of W63”the protection and stabilization of endoglucanase was not observed for both of them, LLCO2 and W63?. I do not undertand the distinction you made.

ü This phrase has been revised and the relation of compound additives and endoglucanase explained more clearly.

38. Line 301. I do not undertand why you mentioned “. T1, T2 and T4 were significantly activated, while T3 and T5 showed slight inhibition on BGL activities of LLC02”. However in the graph 4e it can be seen that the activity of T1, T2 and T4 was lower than the one of LLCO2 (light green bar). The stability was higher. Revise the information because it is confusing.

ü The information results of compound additives of OA and SL on BGL activity of LLC02 and W₆₃ were revised.

39. Line 303. You state “Surprisingly, for BGL activity of W63, no difference in OA species were observed except T3”. Did you calculate significant differences to be sure that values of T3 were different to the rest ?

ü We are agree with the comment. This phrase has been revised.

ü Each sample was evaluated in triplicate, and the results were expressed as the average value.

40. Line 307. Phrase needs revision.

ü We are agree with the comment. This phrase has been revised.

41. Line 308-310. Elaborate more this information

ü We are agree with the comment. This sentence has been revised.

42. Line 313. It should be mentiones that XYL is a hemicellulase

ü We are agree with the comment. Hemicellulase of XYL was added to the description.  

43. Line 315. Replace the comma by a stop.

ü We sincerely thank the reviewer for careful reading. Revised manuscript has been replaced the comma by a stop.

44. Line 314-316. Could you explain this beaviour?

ü We sincerely thank the reviewer’s valuable feedback and revised this information.

45. Line 317. Replace the comma by an “and”.

ü We sincerely thank the reviewer for careful reading. Revised manuscript has been replaced the comma by an “and”.

46. Line 344. I have mentioned it previously. It is very strange you refer to “BGL enzymatic cocktail from W63” when you know that it posses other enzyme activities as we can see in figure 4.

ü We are agree with the comment. This sentence has been revised.

47. Line 346. Specify the enzyme

ü We feel sorry for our carelessness. More information for this sentence was supplement.

48. Specify because C10+S decreased xylanase activity. Revise this information.

ü We sincerely thank the reviewer’s valuable feedback and revised this information.

Reviewer2:

It is impossible to evaluate the changes made to the article, as the quality of the text has deteriorated too much due to English language errors. This article needs to be corrected by a native speaker. I reject this version.

Response:

Dear reviewer,

We apologize for the poor language of our manuscript. We worked on the manuscript for a long time and the repeated addition and removal of sentences and sections obviously led to poor readability. We have now worked on both language and readability and also improved the manuscript with English language editing services. We really hope that the flow and language level have been substantially improved. We sincerely involve the reviewer to comment on our revised manuscript again.

Reviewer3:

I have detected that you have improved some parts of the text, trying to make the work more clear. However, still there are many aspects to be improved. The english must be revised as there are many incomplete phrases and there are phrases that are difficult to understand. The conclusion section also needs to be revised as it is not understandable.

Response

Dear reviewer, thanks for your careful checks. We apologize for the poor language of our manuscript. Based on your comments, we have now worked on both language and readability and also improved the manuscript with English language editing services. We really hope that the flow and language level have been substantially improved.

Question

Response

Line 11-12. I do not understand “cellulase and hemicellulase components, especially for woody biomass”. Cellulase and hemicellulase are enzymes.

ü  We are agree with the comment and this sentence has been revised.

2. Line 12. ¿have received highly concentration?. I would modify this

ü  We are agree with the comment and this sentence has been modified.

3. Line 19-21. This phrase is incomplete

ü  We are agree with the comment and this phrase has been revised.

4. Line 22. What do you mean with facilitating the recovery of cellulase cocktail?

ü  We sincerely thank the reviewer for careful reading. And this sentence has been revised.

5.Line 50-51. Do you know the concentration of these components in this type of biomass?

ü  Yes, poplar has a matrix composed of three major biopolymeric components: lignin (10-21%), hemicellulose (25-28%), and cellulose (45-51%) (Mcintosh et al., 2016)

ü  This was because that CBH and EG act synergistically to degrade native cellulose to cellobiose, which is an inhibitor of both enzymes. In this sences, BGL can hydrolyse the β-1, 4-glycosidic linkage of cellobiose to generate D-glucose. Hence, BGL not only catalyses the final step of cellulose degradation but also can allow the cellulolytic enzymes to function more efficiently by eliminating cellobiose-mediated inhibition. And more details are described in our previous report (Liang et al., 2019.  https://doi.org/10.1007/s10570-019-02477-y)

ü  We are sorry for our carelessness and this sentence has been revised.

ü  We are agree with the comment.

ü  The treated poplar was washed and dried at 60 °C until constant weight

ü  The total working volume was10 mL

ü  We are agree with the comment. And the verb was replaced by the word “addition”.

ü  We sincerely thank the reviewer for careful reading.  

ü  It can be assumed that the compound additive makes it more easy to disperse EG into the CMC substrate.

ü  We are agree with the comment and rewrote this sentence.

ü  We are agree with the comment and rewrote this sentence.

ü  We are agree with the comment and this phrase has been revised.

ü  We are sorry for our carelessness and this phrase has been revised.

ü  It is the enzyme is the enzyme inactivated in the presence of these compounds

ü  We are sorry for our carelessness and this phrase has been revised.

ü  We are studying the single effects of compound additives of T1, T2 and T4.

ü  We are sorry for our carelessness and this word has been revised to “Enhance”

ü  We are agree with the comment and this phrase has been revised.

ü  We are agree with the comment and this phrase has been revised.

ü  We are agree with the comment and this phrase has been revised.

ü  We are agree with the comment and rewrote this phrase.

ü  We are sorry for our carelessness and this phrase has been revised.

ü  We are studying the single effects of compound additives of T1, T2 and T5.

ü  We are sorry for our carelessness and this phrase has been revised.