Lactic Acid Production from Steam-Exploded Sugarcane Bagasse Using Bacillus coagulans DSM2314
Round 1
Reviewer 1 Report
In this study, different enzymatic hydrolysis and fermentation conditions were used to produce LA from sugarcane bagasse after steam explosion at 195 ºC for 7.5 and 15 min. Enzymatic hydrolysis was carried out with Novozymes’ Cellic CTec3 and/or Cellic HTec3, whereas fermentation was performed with Bacillus coagulans DSM2314. Fermentation inhibitors were removed from C5 streams using physical adsorption on activated carbon powder, while fermentation was carried out with Bacillus coagulans DSM2314 using both SHF and SHCF protocols. The final conclusion is that under the conditions of 195 ℃ and 15 minutes, the LA maximum yield of 76.70 g L-1 h-1 can be achieved by using SHCF (separation hydrolysis co fermentation) when C5 detoxification solution is added at a ratio of 10%。However, there are several things that need to be revised and clarified to support the authors’claims.
1. The author needs to explain why physical adsorption was chosen in Introduction.
2. The author had better add a technology roadmap about the different biological processing strategies designed.
3. The author needs to explain in the introduction why the coagulating bacterium DSM2314 is used to produce LA, and what are the advantages of this strain compared to other strains.
4. The author needs to explain the specific range of non-inhibitory concentrations of substances such as acetic acid, furfural, and 5-HMF on fermentation and hydrolysis in Section 2.1.
5. The author needs to add some images to interpretation the experimental background, experimental ideas such us pretreatment techniques or detoxification techniques respectively in the Introduction and Material and Methods.
I have no comments.
Author Response
RESPONSE TO REVIEWER #1
In this study, different enzymatic hydrolysis and fermentation conditions were used to produce LA from sugarcane bagasse after steam explosion at 195 ºC for 7.5 and 15 min. Enzymatic hydrolysis was carried out with Novozymes’ Cellic CTec3 and/or Cellic HTec3, whereas fermentation was performed with Bacillus coagulans DSM2314. Fermentation inhibitors were removed from C5 streams using physical adsorption on activated carbon powder, while fermentation was carried out with Bacillus coagulans DSM2314 using both SHF and SHCF protocols. The final conclusion is that under the conditions of 195 ℃ and 15 minutes, the LA maximum yield of 76.70 g L-1 h-1 can be achieved by using SHCF (separation hydrolysis co fermentation) when C5 detoxification solution is added at a ratio of 10%。However, there are several things that need to be revised and clarified to support the authors’ claims.
- The author needs to explain why physical adsorption was chosen in Introduction.
Answer: Thank you for your comment. Physical adsorption was chosen for simplicity, efficiency, and selectivity toward fermentation inhibitors. Besides, physical adsorption would impart to dilution to the sugar stream and caused no sugar losses after the effective removal of furans derivatives (over 98%) and acetic acid (around 50%). This brought these chemicals to non-inhibitory levels and allowed fermentation to proceed as did the reaction control. A sentence was added to the manuscript to clarify this technical issue (lines 93 to 96).
- The author had better add a technology roadmap about the different biological processing strategies designed.
Answer: Thank you very much for your comment. In our view, the original manuscript already had plenty of information about the technology roadmap, so it was not easy for us to identify changes that could be introduced to satisfy this recommendation for review. The biological processing strategies used in this work are enzymatic hydrolysis and fermentation. Therefore, additional information was added (lines 130 to 133) to emphasize the relevance of our experimental design. Also, and interactive chart was included as Figure 1 in the beginning of Material and Methods to provide a visualization of the complexity and interactiveness of our experimental approach.
- The author needs to explain in the introduction why the coagulating bacterium DSM2314 is used to produce LA, and what are the advantages of this strain compared to other strains.
Answer: Bacillus coagulans DSM2314 was chosen based on its ability to co-ferment pentoses and hexoses to lactic acid. References for this evidence were already cited in the original version of our manuscript submission. Also, Bacillus coagulans DSM2314 seemed more tolerable to fermentation inhibitors. The expected inhibitory levels of acetic acid, furfural, and 5-HMF for several Bacillus strains was already found in our original submission. A new sentence was to state that no information was found in the literature about the tolerance and inhibitory levels of B. coagulans DSM2314 to the chemicals listed above (see lines 103 to 105).
- The author needs to explain the specific range of non-inhibitory concentrations of substances such as acetic acid, furfural, and 5-HMF on fermentation and hydrolysis in Section 2.1.
Answer: The expected inhibitory levels of acetic acid, furfural, and 5-HMF for different Bacillus strains were already given in the original version of our manuscript submission. To the best of our knowledge, there is no direct information out there indicating such inhibitory levels for B. coagulans DSM 2314. This information was included in the Introduction of our revised version (see lines 103 to 105).
- The author needs to add some images to interpretation the experimental background, experimental ideas such us pretreatment techniques or detoxification techniques respectively in the Introduction and Material and Methods.
Answer: To account for this thoughtful comment, a new figure was inserted in the beginning of Material and Methods to provide details about our experimental approach and how the experimental procedures were heavily interactive. Now, Figure 1 gives a clear vision of the complexity and interrelationship of the main activities (chemical characterization, pretreatment, hydrolysis, and fermentation) involved in our work. Besides, a new paragraph was also inserted in the beginning of Material and Methods to introduce Figure 1 and its contents.
Reviewer 2 Report
The presented material seems interesting and requires partial revision.
The authors need to make minor corrections to improve the article.
Comments are provided below.
1. Form a generalized scientific goal of the research, indicate it in the abstract of the article
2. For all raw materials used, indicate the city of origin in addition to the country, as well as the origin of all reagents.
3. Give a more complete description of the research methods
4. In conclusion, it is necessary to indicate possible options for the practical application of the results obtained.
5. The list of references should have sources from 2010-2022
Author Response
RESPONSE TO REVIEWER #2
The presented material seems interesting and requires partial revision. The authors need to make minor corrections to improve the article. Comments are provided below.
- Form a generalized scientific goal of the research, indicate it in the abstract of the article
Answer: Our Abstract contains all information that we could fit on 200 words, and no information could be removed without compromising its technical contents. For this reason, we do not see how the Abstract could be edited to add more information about our scientific goals. Hence, no changes were made due to word count limit imposed by the journal and to the consequences of removing part of its contents in favor of additional (and eventually unnecessary) information about our scientific goals.
- For all raw materials used, indicate the city of origin in addition to the country, as well as the origin of all reagents.
Answer: City of origin and country for all raw materials and reagents are now cited in the revised version of our manuscript. See lines 161, 166, 167/168, 174, 178/179, 218/219, and 227/228 of the revised version.
- Give a more complete description of the research methods
Answer: The specific methodologies for pretreatment, hydrolysis and fermentation were already described in detail in our original submission. On the other hand, all methods used for biomass characterization were based on NREL standard procedures whose details are well described in the literature. For this reason, it seemed not plausible to provide a detailed description for each one of them in Material and Methods. However, the main principle of each analytical procedure was included in revised version of our manuscript, lines 160 to 168.
- In conclusion, it is necessary to indicate possible options for the practical application of the results obtained.
Answer: Our Conclusion was rewritten as requested.
- The list of references should have sources from 2010-2022
Answer: Seven relatively old references (older than 2010) were replaced in the revised version of the manuscript. Some old references could not be removed because they contain important information to the readers. The percentage of old references in our entire reference list dropped from 32% to 22% after manuscript revision.
