Cellulosic Butanol Biorefinery: Production of Biobutanol from High Solid Loadings of Sweet Sorghum Bagasse—Simultaneous Saccharification, Fermentation, and Product Recovery

Round 1

Reviewer 1 Report

The work is interesting, and the results are promising. All objectives are well defined and addressed in the manuscript. The results are fully described and explained, and conclusions are well supported by the results. I have a few minor concerns and suggestions listed below:

Lines 47-48: Why mention a range? You can get the exact number from Annual Industry Outlook: https://ethanolrfa.org/resources/annual-industry-outlook

Lines 84-85: “Use of concentrated solids is not possible until they are saccharified, 84 fermented and product is recovered simultaneously” – I believe authors should elaborate this to make the justification of this work sounder. I am assuming that both substrate and product inhibition can be addressed using this approach, but authors need to clearly mention their hypothesis in the manuscript.

Lines 97-98: As the pretreatment time is zero, it is critical to mention the heating rate. The authors have mentioned the cooling rate but not the heating rate. Also, it would be better if the authors can provide some justification for the selection of this specific temperature.

Lines 169-171: For how long vacuum was applied? What were the time points and on what basis they were selected? Normally, under such conditions, a temperature drop is observed, did authors monitor the temperature in the bioreactor? Also, it would be helpful to provide some details of the bioreactor and temperature-controlled mechanism.

Author Response

We would like to thank the reviewers for their exceptionally good comments that have improved the quality of the manuscript. For our response please see mentioned line numbers in the revised manuscript.

Comment: The work is interesting, and the results are promising. All objectives are well defined and addressed in the manuscript. The results are fully described and explained, and conclusions are well supported by the results. I have a few minor concerns and suggestions listed below: Thank you!

Lines 47-48: Why mention a range? You can get the exact number from Annual Industry Outlook: https://ethanolrfa.org/resources/annual-industry-outlook

Our response: Please see page 2, lines 47-48: This has been changed as suggested by the reviewer 1.

Comment: Lines 84-85: “Use of concentrated solids is not possible until they are saccharified, 84 fermented and product is recovered simultaneously” – I believe authors should elaborate this to make the justification of this work sounder. I am assuming that both substrate and product inhibition can be addressed using this approach, but authors need to clearly mention their hypothesis in the manuscript.

Our response: This is an excellent comment. Looking at lines 73-80 (above paragraph) we have made changes in the following paragraph. Please see lines 83 & 84-86 

Comment: Lines 97-98: As the pretreatment time is zero, it is critical to mention the heating rate. The authors have mentioned the cooling rate but not the heating rate. Also, it would be better if the authors can provide some justification for the selection of this specific temperature.

Our response: The heating rate was 2.6 ^oC.min^-1. It has been included in the revised manuscript. Please see line 107. Also a justification has been given in the Ms. Please see lines 106-107. 

Comment: Lines 169-171: For how long vacuum was applied? What were the time points and on what basis they were selected? Normally, under such conditions, a temperature drop is observed, did authors monitor the temperature in the bioreactor? Also, it would be helpful to provide some details of the bioreactor and temperature-controlled mechanism.

Our response: Vacuum was applied for 30 min each time. Please see line 177. For time points please see Figure 3 A; arrows indicate points when vacuum was applied. Recovery selection basis was ABE concentration of 4.0 or > 4.0 in the reactor (please see lines 180-181). Temperature drop has been mentioned - please see line 178. It was dropped from 35 ^oC to 33.5 ^oC. Details of the bioreactor: it's size was 500 mL (please see line 118). For temperature control the reactor was placed in a water bath - please see lines 178-180. Water bath's details have been provided (lines 178-180). 

Reviewer 2 Report

Reviewer comments to the manuscript entitled “Cellulosic Butanol Biorefinery: Production of Biobutanol from High Solid Loadings of Sweet Sorghum Bagasse - Simultaneous Saccharification, Fermentation, and Product Recovery”, Manuscript Number: 1489864, Fermentation.

The authors studied the consolidated bioconversion and product recovery process of high solid loading SSB to ABE by C. beijerinckii. The authors have an extensive record of research and publications in the field, and the present manuscript is based on a comprehensive research work and an appreciable amount of information. The manuscript is very interesting, innovative, and up-to-date, and in my opinion is acceptable for publication. Nonetheless, there are some discussion topics that could still be covered by the authors and one small correction to be made.

1. How do the authors explain the concentration of 8 g/L acetic acid in the hydrolysate using 160 g/L of SSB solids versus 5.28 g/L using 220 g/L of solids?
2. Can the authors indicate from what concentration acetic acid can be inhibitory for the microorganism?
3. Should the 1.22 g/L of acetic acid used by C. beijerinckii have been considered in the material balances?
4. The losses by diffusion are one important challenge to be overcome. Can the authors propose operational improvements / alternatives to the process scheme that may help to limit those losses?
5. Minor correction: Figure 1 should be revised, as it appears unformatted in the text.

Author Response

We would like to thank the reviewer for his/her excellent comments that have improved the quality of the paper. Our response to his/her comments has been listed below. 

The authors studied the consolidated bioconversion and product recovery process of high solid loading SSB to ABE by C. beijerinckii. The authors have an extensive record of research and publications in the field, and the present manuscript is based on a comprehensive research work and an appreciable amount of information. The manuscript is very interesting, innovative, and up-to-date, and in my opinion is acceptable for publication. Nonetheless, there are some discussion topics that could still be covered by the authors and one small correction to be made.

1. How do the authors explain the concentration of 8 g/L acetic acid in the hydrolysate using 160 g/L of SSB solids versus 5.28 g/L using 220 g/L of solids? Our response: This is an interesting comment. We have provided a response. Please see lines 312-314.
2. Can the authors indicate from what concentration acetic acid can be inhibitory for the microorganism? Our response:  It is expected that the culture can tolerate 10-12 g.L^-1 acetic or butyric acid. Please see lines 323-329. 
3. Should the 1.22 g/L of acetic acid used by C. beijerinckii have been considered in the material balances? Our response: acetic acid was not included in the material balance. Please see line 311-312.
4. The losses by diffusion are one important challenge to be overcome. Can the authors propose operational improvements / alternatives to the process scheme that may help to limit those losses? Our response: there are two ways to avoid any losses: i) use of low condensation temperature, and ii) use of metal, preferably steel, pipes. This has been included in the text. Please see lines 346-349.
5. Minor correction: Figure 1 should be revised, as it appears unformatted in the text. Our response: The figure has been formatted.