Co-Production of Isobutanol and Ethanol from Prairie Grain Starch Using Engineered Saccharomyces cerevisiae
Round 1
Reviewer 1 Report
Overall the manuscript was straight-forward and well-written. It is an interesting manuscript particularly (for me) the use of fungal-contaminated grain. Apart from some minor issues this manuscript can be accepted. In Fig 1, 2, 3 the x-axes showing the time should preferable start where it converges with the y-axes
-line 90: avoid "very" in scientific writing
-line 96: "overexpresses" is normally one word
-line 146: how was this yeast concentration determined?
-line 402: a benefit "of" using
-line 467: than ethanol
Author Response
We thank the reviewer for helpful comments and pointing out several improvements that could be made to the manuscript. All of the suggested changes have been made.
Overall the manuscript was straight-forward and well-written. It is an interesting manuscript particularly (for me) the use of fungal-contaminated grain. Apart from some minor issues this manuscript can be accepted.
(1) In Fig 1, 2, 3 the x-axes showing the time should preferable start where it converges with the y-axes
All graphs have been modified so that the 0 points converge at the x and y axes.
(2) -line 90: avoid "very" in scientific writing
Done, this change has been made as suggested.
(3) -line 96: "overexpresses" is normally one word
Done, this change has been made as suggested.
(4) -line 146: how was this yeast concentration determined?
Done, this change has been made as suggested.
(5) -line 402: a benefit "of" using
Done, this change has been made as suggested.
(6) -line 467: than ethanol
Done, this change has been made as suggested.
Reviewer 2 Report
In my opinion, the captions for Figures 1 and 2 do not completely correspond to the content shown in the diagrams. Why do the Authors focus only on isobutanol production, while the process yields definitely higher ethanol content?
For example, instead of: "Figure 1. Isobutanol production in triticale (squares), wheat (triangles) and barley (circles) mashes" it would be better: "Figure 1. The effects of the SSF process for triticale (squares), wheat (triangles) and barley (circles) mashes.
By analogy, Figure 2 shows not only the increase in isobutanol production, but also ethanol and yeast biomass.
Lines 637-639 is:
Eksteen, J.M.; Van Rensburg, P.; Cordero Otero, R.R.; Pretorius, I.S., Starch fermentation by recombinant saccharomyces cerevisiae strains expressing the alpha-amylase and glucoamylase genes from lipomyces kononenkoae and saccharomycopsis fibuligera. Biotechnol Bioeng, 2003, 84(6): p. 639-646.
should be:
Eksteen, J.M.; Van Rensburg, P.; Cordero Otero, R.R.; Pretorius, I.S., Starch fermentation by recombinant Saccharomyces cerevisiae strains expressing the alpha-amylase and glucoamylase genes from Lipomyces kononenkoae and Saccharomycopsis fibuligera. Biotechnol Bioeng, 2003, 84(6): p. 639-646
Author Response
We thank the reviewer for helpful comments and pointing out several improvements that could be made to the manuscript. All of the suggested changes have been made.
(1) In my opinion, the captions for Figures 1 and 2 do not completely correspond to the content shown in the diagrams. Why do the Authors focus only on isobutanol production, while the process yields definitely higher ethanol content?
-For example, instead of: "Figure 1. Isobutanol production in triticale (squares), wheat (triangles) and barley (circles) mashes" it would be better: "Figure 1. The effects of the SSF process for triticale (squares), wheat (triangles) and barley (circles) mashes.
By analogy, Figure 2 shows not only the increase in isobutanol production, but also ethanol and yeast biomass.
The suggested change has been incorporated into all of the figure legends and isobutanol, ethanol and cell biomass are described.
(2) Eksteen, J.M.; Van Rensburg, P.; Cordero Otero, R.R.; Pretorius, I.S., Starch fermentation by recombinant saccharomyces cerevisiae strains expressing the alpha-amylase and glucoamylase genes from lipomyces kononenkoae and saccharomycopsis fibuligera. Biotechnol Bioeng, 2003, 84(6): p. 639-646.
should be:
Eksteen, J.M.; Van Rensburg, P.; Cordero Otero, R.R.; Pretorius, I.S., Starch fermentation by recombinant Saccharomyces cerevisiae strains expressing the alpha-amylase and glucoamylase genes from Lipomyces kononenkoae and Saccharomycopsis fibuligera. Biotechnol Bioeng, 2003, 84(6): p. 639-646
The title of this reference has been corrected. We thank the reviewer for pointing out this error.
Reviewer 3 Report
The authors in this manuscript have studied isobutanol and ethanol co-production by an engineered yeast using three types of feedstocks. The evaluations include batch fermentation, fed-batch fermentation, effect of nitrogen sources, and effect of mycotoxin (i.g. DON) contaminated grain. The study is technically sound and has scientific value to the overall biofuel research area. The manuscript is well written in English. However, some revisions are needed before publishing in the journal of Fermentation. Some comments are listed below for consideration.
Materials and Methods
P3 L111-113, Are these commercial products? Please give full name for "AC" when it first appears.
P3 L118, This should be expressed based on the format: equipment (model, manufacturer, city, country)
P3 L119, How about the temperature, room T?
P3 L122, “Gencor”, Is this a company? Use format: company (city, country)
P3 L123, “OPTIMASH™”, Is this enzyme commercial? If so please mention the supplier and manufacturer.
P3 L125, “FERMGEN”, Please provide information of supplier and manufacturer.
P3 L127-130, Same as above. Are these enzymes made in the lab or from commercial? Please provide information.
Section 2.4, Have you provided the methods for fed-batch fermentation, as there are results and discussion in section 3.3?
Results
P5 L185-191, This part should be moved to Introduction section.
Figures, Please be consistent for the figure format. As Figure 1 has legends which make the figure easier to read, please do the same for Figure 2 and Figure 3.
Table 2, Is there a control treatment without addition of these nitrogen source? it would give direct information as how effective when dosing each nitrogen source.
P9 L360, “fungal growth”, The reduction of glucose and xylose in Barly FHB is not remarkable compared to clean Barley, thus a conclusion of due to fungal growth is not strong. It is highly suggested to evaluate the fungal biomass in both type of Barley mashes such as measuring glucosamine in fungal cell wall to indirectly determine the the fungal growth in the feedstock.
P10 L366, What is statistical analysis method was used to determine the significant difference? Please add in the Materials and Methods section. Also, what p value was used (i.e. 0.05 or 0.01) for the statistical analysis?
Discussion
P11 L416-424, A comparison of the engineered yeast strain used in this study and those in the literatures would strengthen the discussion here, especially what was different of this strain from other engineered strains in improving butanol production.
Author Response
We thank the reviewer for thoughtful evaluation, helpful comments and pointing out several improvements and relevant additions that could be made to the manuscript. All of the suggested changes have been made.
(1) P3 L111-113, Are these commercial products? Please give full name for "AC" when it first appears.
AC is part of the name of the strain of grain and stands for Agriculture Canada, the agency that developed the strain. This is now indicated in the manuscript (line 121-122)
(2) P3 L118, This should be expressed based on the format: equipment (model, manufacturer, city, country)
This correction has been made as suggested and now reads “Retsch mill (model ZM 100, Retsch Gmbh & Co., Haan Germany)” (line 130)
(3) P3 L119, How about the temperature, room T?
We now indicate that samples were stored at 4 degrees C (line 131).
(4) P3 L122, “Gencor”, Is this a company? Use format: company (city, country)
Genecor is a company and this has been corrected to read “Genecor International (Palo Alto, CA)” (line 135)
(5) P3 L123, “OPTIMASH™”, Is this enzyme commercial? If so please mention the supplier and manufacturer.
All enzymes used are commercially available and this now reads “All enzymes used in this study are commercially available and were produced by and obtained from Genecor International (Palo Alto, CA)”
(6) P3 L125, “FERMGEN”, Please provide information of supplier and manufacturer.
All enzymes used are commercially available and this now reads “All enzymes used in this study are commercially available and were produced by and obtained from Genecor International (Palo Alto, CA)”
(7) P3 L127-130, Same as above. Are these enzymes made in the lab or from commercial? Please provide information.
All enzymes used are commercially available and this now reads “All enzymes used in this study are commercially available and were produced by and obtained from Genecor International (Palo Alto, CA)”
(8) Section 2.4, Have you provided the methods for fed-batch fermentation, as there are results and discussion in section 3.3?
Methods describing the fed-batch process are now included under section 2.5 (line186 – 191).
(9) P5 L185-191, This part should be moved to Introduction section.
We agree, the suggested lines have been integrated into the introduction as (lines 79 – 84).
(10) Figures, Please be consistent for the figure format. As Figure 1 has legends which make the figure easier to read, please do the same for Figure 2 and Figure 3.
We agree, the figures have been changed so that all display a similar legend scheme that makes the figures easier to interpret.
(11) Table 2, Is there a control treatment without addition of these nitrogen source? it would give direct information as how effective when dosing each nitrogen source.
We have added the requested data to Table 2, an d it is described (lines 408-410). It is a helpful addition that adds some context to the effect of added nitrogen sources and highlights the need for added nitrogen to achieve effective fermentation of the available carbon.
(12) P9 L360, “fungal growth”, The reduction of glucose and xylose in Barly FHB is not remarkable compared to clean Barley, thus a conclusion of due to fungal growth is not strong. It is highly suggested to evaluate the fungal biomass in both type of Barley mashes such as measuring glucosamine in fungal cell wall to indirectly determine the the fungal growth in the feedstock.
We agree with this point, the statement that the reduced sugar in mashes from FHB infected grain was an over interpretation. Indeed as the reviewer has pointed out the difference in glucose concentration is relatively small and is in fact not statistically significant. This line now reads “The glucose yield from the F. graminearum contaminated grain was lower than from the clean grain but the difference was not statistically significant (Table 3)”. As suggested by the reviewer we have performed an analysis of the starch samples to determine the presence of fungus infection. Mycological analysis was performed on triplicate samples to detect the presence of fungus. This approach allowed the detection of both actively growing fungus and fungal spores. The analysis revealed significant fungal contamination of the infected grain ~ 80000 CFU/g vs the uninfected grain ~670 CFU/g (lines 458 - 461). While this provides only a relative estimate of the degree of fungal contamination it is among the most widely used procedures for this purpose and the goal of the experiment is simply to determine if grain infected with fungus can be used as an effective feedstock for ethanol and isobutanol synthesis. Additionally, the methodology for performing this analysis is included as a new section 2.6 in the methods.
(13) P10 L366, What is statistical analysis method was used to determine the significant difference? Please add in the Materials and Methods section. Also, what p value was used (i.e. 0.05 or 0.01) for the statistical analysis?
Statistical analysis of all data was performed using a two tailed t-test accepting a p value <0.05 as significant. This information is provided in the methods section 2.7 (lines 225 - 228).
(14) P11 L416-424, A comparison of the engineered yeast strain used in this study and those in the literatures would strengthen the discussion here, especially what was different of this strain from other engineered strains in improving butanol production.
We have added a section to the discussion specifically describing the similarities and differences between our strain and others used for isobutanol biosynthesis and have made reference to titres and yields of isobutanol produced by other strains (lines 537-546). We acknowledge that our strain is not the highest producer of isobutanol but this does not detract from the analysis that has been performed and the relevance of our strain being an effective producer of both isobutanol and ethanol.
Again we thank the reviewer for the helpful suggestions to improve our man uscript.
Round 2
Reviewer 3 Report
The authors have properly addressed most of the comments raised during the first revision. Therefore, it is recommended that the manuscript being published in the journal Fermentation.
