Enhancement of Liquid Hot Water Pretreatment on Corn Stover with Ball Milling to Improve Total Sugar Yields
Round 1
Reviewer 1 Report
Comments and Suggestions for AuthorsA classic and well-done study of pretreatment. Hot-water treatment followed by ball milling has been studied previously, however, I do not remember seeing the use of ball milling before hot-water treatment. While I cannot comment on the economics, I think is an interesting idea that advances knowledge in this field.
Consider modifying title: Enhancement of liquid hot-water pretreatment of corn stover by ball milling.
Abstract
Line 21: lignin redeposition on cellulose surface – please, remove unless visual evidence is provided here.
Line 27-29: please rephase. The point is not that there is a scarcity of petroleum; the point is that because of climate change, we cannot afford to use all the available deposits.
Line 29: Corn is grown on approximately 160 million. Corn stover is currently left on the fields to decompose and is therefore a promising feedstock for production of biofuels and chemicals.
Line 27 – 37: assume reader knows about the need for enzymatic digestion following pretreatment; please state explicitly.
39: have should be has
38-49: please introduce the concept of porosity, which is more important than particle size.
51-52: liquid hot-water is not low in capital costs, just lower than dilute acid or alkaline based processes.
117: think you mean that enzymatic digestions were conducted in 50 mM citrate buffer
124: is it supposed to be 5 mg/g bioimass?
Figure 1: anyway to include names directly on legend rather than A – E? Perhaps, shrink graph a little bit?
223: might not be long enough..
236: start new paragraph with Enhancement
241: start new paragraph with “All three combined pretreatments
254: reported the highest observed glucose yield
Figure 4: I really like this figure! Please, prepare a similar figure for xylose yield.
326: unless you report specific energy consumption for LHW and Ball Milling, it is not possible to suggest that ball milling followed by LHW save energy. I also disagree with the conclusion regarding drying in as far as ball missing con be conducted on wet samples. You can simply state the importance of savings on enzyme costs and increased sugar yields and suggest that energy calculations still need to be carried out.
341: please delete comment about lignin re-distribution unless electron microscopy was conducted in this study.
Future study: might be interesting to see if adding a protein blocker of surfactant would improve the results for the 3 FPU cellulase tests.
Future suggestion: I think it makes more commercial sense to run the whole hydrolysate versus the washed solids because it saves on process water and unit operations, albeit not sure if Ctec2 has good amount of xylanase activity.
Comments on the Quality of English Language
The manuscript is quite understandable as written and generally well written. A moderate amount of polishing would improve the overall style.
Author Response
Response to Reviewer #1
Comment 1: A classic and well-done study of pretreatment. Hot-water treatment followed by ball milling has been studied previously, however, I do not remember seeing the use of ball milling before hot-water treatment. While I cannot comment on the economics, I think is an interesting idea that advances knowledge in this field.
Response 1: Thank you for your valuable comments. Ball milling and liquid hot water pretreatment are both environment friendly process without chemicals. In this study, we combined short-duration ball milling followed by liquid hot water pretreatment under mild conditions on corn stover to achieve both high glucose and xylose yields compared to ball milling and liquid hot water pretreatment alone.
Comment 2: Consider modifying title: Enhancement of liquid hot-water pretreatment of corn stover by ball milling.
Response 2: Agree. The title of manuscript has been revised as “Enhancement of liquid hot water pretreatment on corn stover by ball milling to improve total sugar yields”.
Comment 3: Line 21: lignin redeposition on cellulose surface – please, remove unless visual evidence is provided here.
Response 3: Agree. The expression “lignin redeposition on cellulose surface” has been removed in the revised manuscript.
Comment 4: Line 27-29: please rephase. The point is not that there is a scarcity of petroleum; the point is that because of climate change, we cannot afford to use all the available deposits.
Response 4: Agree. The expression has been modified as “Lignocellulosic biomass has gained considerable attention as a promising and renewable resource for fuel production. This interest has been driven by growing concerns about cli-mate change resulting from the excessive use of fossil energy.” in Line 26-28 the revised manuscript.
Comment 5: Line 29: Corn is grown on approximately 160 million. Corn stover is currently left on the fields to decompose and is therefore a promising feedstock for production of biofuels and chemicals.
Response 5: Agree. Thee expression has been modified as “Corn is grown on approximately 160 million and is currently left on the fields to decom-pose, making it a promising feedstock for the production of biofuels and chemicals.” in Line 28-30 in the revised manuscript.
Comment 6: Line 27 – 37: assume reader knows about the need for enzymatic digestion following pretreatment; please state explicitly.
Response 6: Agree.
(1) The necessity for pretreatment before enzymatic digestion has been elaborated in the line 30-40 in the revised manuscript.
(2) The conversion process of lignocellulosic biomass to bioethanol primarily involves four steps: pretreatment, enzymatic hydrolysis of cellulose and hemicellulose into monosac-charides, fermentation of monosaccharides into ethanol, and the subsequent separation and purification of bioethanol. Lignocellulosic biomass primarily comprises cellulose, hemicellulose, and lignin, which are interconnected. The complexity of these components contributes to biomass recalcitrance, leading to low efficiency and high costs in the con-version of bioethanol. Therefore, pretreatment of lignocellulosic biomass is crucial to modify its structure and enhance the accessibility of cellulose and hemicellulose. Various pretreatments have been utilized to enhance sugar yields of lignocellulosic biomass, such as mechanical grinding, liquid hot water, acid and alkaline pretreatments
Comment 7: 39: have should be has
Response 7: Agree. “have” has been modified as “has” in Line 42 in the revised manuscript.
Comment 8: 38-49: please introduce the concept of porosity, which is more important than particle size.
Response 8: Agree. The concept of porosity has been introduced in line 42-44 in the revised manuscript.
Comment 9:51-52: liquid hot-water is not low in capital costs, just lower than dilute acid or alkaline based processes.
Response 9: Agree. The expression has been modified in Line 56-57 in the revised the revised manuscript.
Comment 10:117: think you mean that enzymatic digestions were conducted in 50 mM citrate buffer
Response 10: Agree. The concentration of citrate buffer has been modified in Line 132 in the revised manuscript.
Comment 11: 124: is it supposed to be 5 mg/g bioimass?
Response 11: Agree. The unit of enzyme addition has been modified in line 139 in the revised manuscript.
Comment 12:Figure 1: anyway to include names directly on legend rather than A – E? Perhaps, shrink graph a little bit?
Response 12: Agree. The Figure 1 has been modified in the revised manuscript. The names are directly on legend instead of A-E in the revised Figure 1.
Comment 13:223: might not be long enough..
Response 13: Agree. The expression has been modified in Line 220-221 in the revised manuscript.
Comment 14:236: start new paragraph with Enhancement
Response 14: Agree. The new paragraph suggested by reviewer has been modified in Line 235 in the revised manuscript.
Comment 15:241: start new paragraph with “All three combined pretreatments
Response 15: Agree. The new paragraph suggested by reviewer has been modified in Line 241 in the revised manuscript.
Comment 16: 254: reported the highest observed glucose yield
Response 16: Agree. The word “yield” has been added in Line 253 in the revised manuscript.
Comment 17: Figure 4: I really like this figure! Please, prepare a similar figure for xylose yield.
Response 17: Agree. A similar figure (Figure 5) for xylose yield has been added in the revised manuscript. And the detailed description of this figure has been added in Line 272-279 in revised manuscript.
Comment 18:326: unless you report specific energy consumption for LHW and Ball Milling, it is not possible to suggest that ball milling followed by LHW save energy. I also disagree with the conclusion regarding drying in as far as ball missing con be conducted on wet samples. You can simply state the importance of savings on enzyme costs and increased sugar yields and suggest that energy calculations still need to be carried out.
Response 18: Agree.
(1) The expression has been modified in Line 318-3324 in the revised manuscript.
(2) The total sugar yield (42.9%) of BM20LHW190 at 3 FPU/g was higher than observed for BM60 with 20 FPU/g (42.7%), indicating that combining ball milling for 20 min with liquid hot water pretreatment at 190°C could save 67% ball milling time and 85% enzyme cost based on comparable total sugar yield. Our previous study demonstrated that as the ball milling time increased from 30 minutes to 60 minutes, the energy requirement during the mechanical fragmentation process doubled. It can be inferred that the combined pretreatment of ball milling and liquid hot water has the potential to reduce energy costs compared to ball milling pretreatment alone. However, detailed energy calculations need to be carried out in the future to confirm this hypothesis.
Comment 19:341: please delete comment about lignin re-distribution unless electron microscopy was conducted in this study.
Response 19: Agree. The expression about lignin re-distribution has been deleted in the revised manuscript.
Comment 20: Future study: might be interesting to see if adding a protein blocker of surfactant would improve the results for the 3 FPU cellulase tests.
Response 20: Agree. In fact, we have already employed bovine serum albumin (BSA) as a surfactant protein blocker to enhance the glucose yield at 3 FPU. The results are illustrated in the figure below. However, it appears that the enhancement in enzymatic hydrolysis was not statistically significant. As a result, we have decided not to include these findings in the manuscript. Thank you again for your valuable comments.
Comment 21:Future suggestion: I think it makes more commercial sense to run the whole hydrolysate versus the washed solids because it saves on process water and unit operations, albeit not sure if Ctec2 has good amount of xylanase activity.
Response 21: Thank you for your valuable comments. We agree with you that running the whole hydrolysate instead of washed solids can have commercial benefits, such as saving on process water and reducing the number of unit operations. Conducting a thorough analysis and feasibility study in future will help determine the most optimal approach.
Author Response File: 
Author Response.pdf
Reviewer 2 Report
Comments and Suggestions for AuthorsGenerally the paper is well-written and the conducted experiments are well-described. I suggest to improve the Introduction section. In my opinion, new findings and new contribution is not clearly emphasized in the Introduction section. Can the authors improve this part? For example, a similar study can be found in [30].
[30] Kim, S. M., Dien, B. S., Tumbleson, M. E., Rausch, K. D., & Singh, V. (2016). Improvement of sugar yields from corn stover using sequential hot water pretreatment and disk milling. Bioresource Technology, 216, 706-713.
I have also some minor remarks. When reading the paper I could find some typographical and grammatical errors. Some examples:
H2SO4 --> H2SO4
log R0 --> log R0
"xylan has been total removed" --> "xylan has been completely removed"
"Most of the lager fractions" --> "Most of the larger fractions"
The paper is well-written and I have only some minor comments that were specified previously.
Author Response
Response to Reviewer #2
Comment 1: Generally the paper is well-written and the conducted experiments are well-described. I suggest to improve the Introduction section. In my opinion, new findings and new contribution is not clearly emphasized in the Introduction section. Can the authors improve this part? For example, a similar study can be found in [30].
[30] Kim, S. M., Dien, B. S., Tumbleson, M. E., Rausch, K. D., & Singh, V. (2016). Improvement of sugar yields from corn stover using sequential hot water pretreatment and disk milling. Bioresource Technology, 216, 706-713.
Response 1: Agree. Thank you for your valuable comments.
(1) The reference by Kim, S. M. has been elaborated in Introduction section in the revised manuscript.
(2) The Introduction section has been improved in the revised manuscript.
(3) Recently, there has been a notable increase in attention towards the integration of physicochemical or chemical pretreatment with physical pretreatment methods. These combined pretreatment approaches are capable of effectively overcoming the limitations of individual pretreatment methods while amplifying their respective advantages. To overcome the shortcoming of ball milling pretreatment and liquid hot water pretreatment, combined pretreatment was also studied. Kim et al. proposed hot water pretreatment at 160–200°C for 4–8 min followed by disk milling, resulting in an improvement of glucose and xylose yields by 89% and 134%, respectively. However, the sequential application of LHWP followed by milling has a significant drawback in terms of its high energy de-mand for the water drying process. Therefore in this study, we combined short-duration ball milling followed by liquid hot water pretreatment under mild conditions on corn stover to achieve both high glucose and xylose yields compared to ball milling and liquid hot water pretreatment alone.
Comment 2:H2SO4 --> H2SO4
Response 2: Agree. H2SO4 has been modified as H2SO4 in Line 144 in the revised manuscript.
Comment 3:log R0 --> log R0
Response 3: Agree. log R0 has been modified as log R0 in Line 168, 170, 178, 180 in the revised manuscript.
Comment 4:"xylan has been total removed" --> "xylan has been completely removed"
Response 4: Agree. The expression has been modified in Line 171 in in the revised manuscript.
Comment 5:"Most of the lager fractions" --> "Most of the larger fractions"
Response 5: Agree. The expression has been modified in Line 207 in in the revised manuscript.
Author Response File: Author Response.pdf
Reviewer 3 Report
Comments and Suggestions for AuthorsThe manuscript (sustainability-2725645) combined ball milling followed by liquid hot water pre-treatment of corn stover to improve high glucose and xylose yields. The manuscript was well organized for possible publication. My detailed comments are listed below.
1. To reveal the changes of crystalline structures before and after various pretreatments, the corresponding WAXD data is desired to better understand the roles of ball milling and/or liquid hot water pre-treatment on sample physical structures. Besides yields, it would be better to further analysis the detailed effects of different pre-treatments or their combinations on the structures.
2. As compared with conventional methods, could authors comment more on energy saving based on the proposed method?
3. Comparing LHW210 and BMLHW190, besides initial hydrolysis rate, are there other disadvantages?
Author Response
Response to Reviewer #3
The manuscript (sustainability-2725645) combined ball milling followed by liquid hot water pre-treatment of corn stover to improve high glucose and xylose yields. The manuscript was well organized for possible publication. My detailed comments are listed below.
Comment 1: To reveal the changes of crystalline structures before and after various pretreatments, the corresponding WAXD data is desired to better understand the roles of ball milling and/or liquid hot water pre-treatment on sample physical structures. Besides yields, it would be better to further analysis the detailed effects of different pre-treatments or their combinations on the structures.
Response 1: Agree.
(1) Many pretreatment methods have an impact on the crystalline structure of cellulose, resulting in altering the enzymatic digestibility of cellulose. In our previous study (Bioresource Technology, 2017, 241, 262-268.), we have utilized X-ray diffraction analysis and solid-state CP/MAS 13C NMR to obtain the crystalline structures, the results are as following. It could be concluded that the crystalline structure of cellulose gradually gets disrupted, resulting in a decrease in crystallinity with increase of ball milling time.
(2) In this study, we focused on the effects of combined pretreatment on chemical composition, particle size distribution and total sugar yield at low and high enzyme loading. In future studies, we will pay more attention to the structural changes brought about by this combined pretreatment approach.
Related reference:
[1] Guanya Ji, Lujia Han, Chongfeng Gao, Weihua Xiao, Yang Zhang, Yaoyao Cao. Quantitative approaches for illustrating correlations among the mechanical fragmentation scales, crystallinity and enzymatic hydrolysis glucose yield of rice straw. Bioresource Technology, 2017, 241, 262-268.
[2] Guanya Ji, Chongfeng Gao, Weihua Xiao, Lujia Han. Mechanical fragmentation of corncob at different plant scales: Impact and mechanism on microstructure features and enzymatic hydrolysis. Bioresource Technology, 2016, 205, 159-165.
Comment 2: As compared with conventional methods, could authors comment more on energy saving based on the proposed method?
Response 2: Agree.
(1) The energy analysis has been modified in Line 316-325 in the revised manuscript.
(2) The total sugar yield (42.9%) of BM20LHW190 at 3 FPU/g was higher than observed for BM60 with 20 FPU/g (42.7%), indicating that combining ball milling for 20 min with liquid hot water pretreatment at 190°C could save 67% ball milling time and 85% enzyme cost based on comparable total sugar yield. Our previous study demonstrated that as the ball milling time increased from 30 minutes to 60 minutes, the energy requirement during the mechanical fragmentation process doubled. It can be inferred that the combined pretreatment of ball milling and liquid hot water has the potential to reduce energy costs compared to ball milling pretreatment alone. However, detailed energy calculations need to be carried out in the future to confirm this hypothesis.
(3) Compared to traditional acid or alkaline based processes, the main advantage of the proposed approach in this study may not be energy savings, but rather the absence of chemical reagents, making it environmentally friendly.
Comment 3: Comparing LHW210 and BMLHW190, besides initial hydrolysis rate, are there other disadvantages?
Response 3: Agree.
(1) The comparison between LHW210 and BMLHW190 in terms of glucose yield primarily focuses on the initial hydrolysis rate. However, when considering xylose yield, the drawbacks of LHW210 are significant. To elaborate this point, we have added a new figure (72h Xlycose yield of enzymatic hydrolysis for all samples.) in the revised manuscript.
(2) Liquid hot water pretreatment at low severity enhanced the xylose yield. However, at high severity, the liquid hot water pretreatment resulted in the destruction of xylan components in corn stover, leading to zero xylose yield. The highest xylose yield was achieved in the combined pretreatment samples.
Author Response File: Author Response.pdf
