Dynamic Co-Cultivation Process of Corynebacterium glutamicum Strains for the Fermentative Production of Riboflavin
, Arthur Burgardt 2, Dina R. Kallman 1, Volker F. Wendisch 2 and Nadav Bar 1
Round 1
Reviewer 1 Report
Dear authors,
The paper described a very deep study in the production of riboflavin using Corynebacterium glutamicum. In my opinion, the paper is very well written, everything explain in detail, being easy to follow in spite all the included data. However, I will suggest to do some modifications which are detailed below:
Page , line 40. Space before “although”
Line 41. I think you mean “the raw material composition”
Line 55. Include tipical sources of vitamin B2 in food
Line 55. It should be included that it can be incorporated through supplements
Line 77. What is the difference of both C. glutamicum strains?
Line 107. In the SynSSL, which concentrations of the three sugars were used?
Line 111. Include autoclave conditions
Line 148. Space after pH
Line 161. Why was phosforic acid used to control the pH?
Line 170. 0.2 mg biotin
Line 177. It is mentioned that linear feeding rate of 0.4 mL min-1 was initiated when glucose was depleted. Was this glucose measured by HPLC?
Line 198. Which was the glucose concentration to obtain the values of 0.25 ± 0.01 h-1 and 0.40 ± 0.02 g g-1, 5, 10 or 20 g L-1?
Line 199. I do not undertand the phrase
Why experiments in Figure 2 were not repited?. How do you know they are reproducible?
Line 341. Detail the information given in the phrase “ The sum of the by-products remaining in the supernatant…”
Line 407. Why mannose and xylose were not consumed?
After obtaining such good results with the synthetic SSL, why didn´t you try with the real spent sulfite liquors?
Author Response
Thank you for your thoughtful input. Please find below the answers to your questions and requests. Line numbers refer to the revised manuscript.
Page , line 40. Space before “although”
Corrected (line 41).
Line 41. I think you mean “the raw material composition”
Corrected (line 42).
Line 55. Include tipical sources of vitamin B2 in food
The information has been added to the manuscript as requested (line 56).
Line 55. It should be included that it can be incorporated through supplements
The information has been added to the manuscript as requested (line 57).
Line 77. What is the difference of both C. glutamicum strains?
The strain C. glutamicum DM1800 overproduces l-lysine, while the strain C. glutamicum ∆lysA is l-lysine auxotrophic
The information has been added to the manuscript (line 82 – 83).
Line 107. In the SynSSL, which concentrations of the three sugars were used?
The synSSL used during growth experiments on flasks contained a mixture of 5, 10, 20, 50 or 100 g L-1 per each sugar. The synSSL used during bioreactor experiments contained a mixture 20 g L-1 per each sugar.
The information has been added to the manuscript (line 112 – line 115).
Line 111. Include autoclave conditions
The information has been added to the manuscript as requested (line 118)
Line 148. Space after pH
Corrected (line 155).
Line 161. Why was phosforic acid used to control the pH?
The metodology for the fed-batch fermentations applied in our manuscript is based on previous works (Henke et al., 2017, Mindt et al., 2018, Perez-Garcia et al., 2018). In such works, successful fed-batch fermentations with C. glutamicum strains were performed using phosphoric acid 10% (v/v) to adjust the pH. However, other fed-batch processes with C. glutamicum used sulfuric acid instead (Blombach et al., 2011; Wieschalka et al., 2012). Phosphoric acid is a weaker acid compared to sulfuric acid but sufficiently strong to control the pH without damaging equipment and plastic hoses during long fermentations.
Line 170. 0.2 mg biotin
Corrected (line 177).
Line 177. It is mentioned that linear feeding rate of 0.4 mL min-1 was initiated when glucose was depleted. Was this glucose measured by HPLC?
Yes, sugar concentrations were monitored at-line continuously along the fed-batch processes.
For more clarity, the text has been modified (line 184; line 415).
Line 198. Which was the glucose concentration to obtain the values of 0.25 ± 0.01 h-1 and 0.40 ± 0.02 g g-1, 5, 10 or 20 g L-1?
The values showed in this sentence are the average and standard deviation of the values obtained from 5, 10 or 20 g L-1 of glucose.
For more clarify, the text has been modified (line 205 – 207).
Line 199. I do not undertand the phrase
The sentence has been modified for more clarity (line 208 – 209).
Why experiments in Figure 2 were not repited?. How do you know they are reproducible?
We did not repeat the experiments in Figure 2 (as indicated in the legend to figure 2 in the original as well as in the revised manuscript) because we considered that experiments performed in bioreactor are tightly monitored and controlled leaving little space for variations. Moreover, the riboflavin production values obtained in the bioreactor experiments confirmed our shake flask experiments. This last sentence has been added to the manuscript (line 318 - 319).
Line 341. Detail the information given in the phrase “ The sum of the by-products remaining in the supernatant…”
The sentence has been modified to add more details as requested by the reviewer (line 355).
Line 407. Why mannose and xylose were not consumed?
As the reviewer observed correctly, by the end of the fed-batch processes (when pO2 raised from 30% to above 50%) the sugars xylose and mannose were not fully depleted. We can only assume that either the cells were metabolically exhausted due to the long fermentation process and/or that the medium used is not optimal for the tested conditions and, therefore, some essential nutrients could have been consumed.
This information has been added to the discussion of the manuscript (line 572 – 576).
After obtaining such good results with the synthetic SSL, why didn´t you try with the real spent sulfite liquors?
As the reviewer pointed out, the next logic step within this research line would be fermentations with real SSL. In this study, we focused only on the consumption of the main sugar components and not on the resilience of the strains to the inhibitors. In the future, we plan to address sugar consumption in the presence of growth inhibitors that can be considered the most important difference between SSL and synSSL. Inhibitors in SSL are, for example, acetic acid, furfural and 5-hydroxymethylfurfural (Rueda et al., 2015). Naturally, C. glutamicum can utilize acetic acid for growth and production (Henke et al., 2019). Under oxygen-deprivation conditions, C. glutamicum has a better recalcitrance towards furfural and 5-hydroxymethylfurfural than E. coli and S. cerevsiae (Tsuge et al 2014), but aerobic growth, as studied here, is negatively affected. These limitations can be overcome in upcoming projects via adaptive laboratory evolution as shown in C. glutamicum for methanol or indole (Hennig et al., 2020, Walter et al., 2020) or by genetic engineering as shown for by furfural detoxification by protein FudC (Tsuge et al., 2016). We believe this work is important for application of real SSL, but not within the scope of the study presented here.
This information has been added to the discussion of the manuscript (line 604 - 613).
Reviewer 2 Report
Reviewer’s Comments
Title: Dynamic co-cultivation process of Corynebacterium glutamicum strains for the fermentative production of riboflavin.
Manuscript ID: fermentation-1042891
Authors: Perez-Garcia et al.
Decision: Accept with minor corrections
Overview: The manuscript describes production of riboflavin from synthetic spent sulfite liquors (SSLs) through overexpression of sugar metabolic pathways in Corynebacterium glutamicum. The study is interesting and the developed C. glutamicum strains provides an economic means of riboflavin production. The manuscript is well written; however, it contained a few grammatical errors that should be addressed. The authors should also address the following concerns:
- Information on the range of microbial inhibitors and sulfites in SSLs should be provided. Also, a synthetic SSL medium that includes the range of inhibitors and sulfites should be used to test glutamicum strains.
- Lines 197-199: Does the sentence mean that RiboGlu strain supplemented with 5, 10, 20 g/L glucose had the same growth rate and yield?
- The sentence ‘For the same concentrations of glucose, the riboflavin yields were 0.22±0.02 mg g-1’ is hard to follow. It will be nice if it is revised.
- Figure 1. What strain is used as the control? Please provide a control strain and compare the developed strains to the control.
- Line 389: How many independent fed-batch operations were carried out?
Author Response
Thanks for your comments. The manuscript has been carefully reviewed. Please find below the answers to your questions and requests. Line numbers refer to the revised manuscript:
- Information on the range of microbial inhibitors and sulfites in SSLs should be provided. Also, a synthetic SSL medium that includes the range of inhibitors and sulfites should be used to test glutamicum strains.
As requested by the reviewer, a more detailed description of the SSL composition was provided in the manuscript (line 39 – 41).
In this work we focused only on the consumption of the main sugar components and not on the resilience of the strains to the inhibitors. That is the main reason for using synSSL instead of real SSL. Upcoming research projects will focus on real SSL and will investigate in detail the effect of these growth inhibitors on growth and production.
Regarding this topic, new information has been added to the discussion (line 604 - 613).
- Lines 197-199: Does the sentence mean that RiboGlu strain supplemented with 5, 10, 20 g/L glucose had the same growth rate and yield?
The values showed here represent the average and standard deviation of the values obtained from 5, 10 or 20 g L-1 of glucose.
For more clarify, the sentence has been modified (line 205 – 207).
- The sentence ‘For the same concentrations of glucose, the riboflavin yields were 0.22±0.02 mg g-1’ is hard to follow. It will be nice if it is revised.
As requested, the sentence has been rephrased (line 208 – 209).
- Figure 1. What strain is used as the control? Please provide a control strain and compare the developed strains to the control.
In this set of experiments, the strain RiboGlu growing on glucose was considered as control since it is the strain not harboring any modification related to carbon source consumption. This was clarified in the manuscript (line 202 – 203).
In addition, the comparison between RiboGlu and the other strains has been improved in the manuscript (line 212 – 213; line 215 –216; line 220 – 221).
- Line 389: How many independent fed-batch operations were carried out?
Two independent fed-batch operations were carried out. One process was performed with the strain RiboSSL while the other process was performed with the strains RiboMan and RiboXyl in dynamic co-cultivation.
This information was clarified in the manuscript (Line 404 – line 405).
