Design-of-Experiment-Guided Establishment of a Fermentative Bioprocess for Biomass-Bound Astaxanthin with Corynebacterium glutamicum

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

This work optimized the fermentation conditions of astaxanthin production by Corynebacterium glutamicum engineered strain. The astaxanthin production reached 64 mg/L through the fed-batch process. This work has a guiding significance for industrial fermentation production of astaxanthin, but there are still some problems that need to be addressed.

1. The formats of all tables are not uniform, especially the table lines.

2. Table1: How are the levels of each factor in the table determined? For example, why did rDOS choose 15%, 30%, and 45%?

3. rDOS and aeration rate are obviously two highly correlated factors. Was the interaction between these two factors considered in the experiment?

4. Figure 3-5: Are there any repeated experiments and statistical analysis, otherwise how can the optimization results be explained?

Author Response

This work optimized the fermentation conditions of astaxanthin production by Corynebacterium glutamicum engineered strain. The astaxanthin production reached 64 mg/L through the fed-batch process. This work has a guiding significance for industrial fermentation production of astaxanthin, but there are still some problems that need to be addressed.

The formats of all tables are not uniform, especially the table lines.

-_-> We thank the Reviewer for this mindful advice and harmonized the layout of all tables in the manuscript. Only for Table 3 we kept some additional internal lines to highlight subheadings within the table.

Table1: How are the levels of each factor in the table determined? For example, why did rDOS choose 15%, 30%, and 45%?

-_-> We added an additional clarification for the setpoints at the end of the introduction and to the description of table 1. The default pO2 setpoint of 30% was chosen as it is a quite common level for fermentation and was also previously used by our research group for astaxanthin fermentation (e.g. DOI:10.3390/molecules28041996). It allows both sufficient oxygen supply while also limiting the required agitation rates. The value was then varied by 50% to have enough of a difference between the 3 levels and for the design it is required that the relative distance between the levels is the same. Similar considerations were responsible for the choice in aeration rate. The default pH was set to 7 as it is the usual pH value for Corynebacterium glutamicum growth. The different levels chosen varied by 1 pH unit to give enough of a range to observe differences while still being inside the viable pH range of C. glutamicum. For the aeration rate previous works have typically used 0.5 – 1 vvm (e.g. DOI:10.3389/fbioe.2020.630476, DOI:10.3390/molecules28041996) so we tested reducing this even further. Similar considerations were used for the initial OD.

rDOS and aeration rate are obviously two highly correlated factors. Was the interaction between these two factors considered in the experiment? 

-_-> The initial DoE approach accounts for two-factor interactions, although these are aliased with each other due to the experimental design. For astaxanthin we observed no statistically significant two-factor interactions at all, while for the total carotenoids we observed a significant two-factor interaction from either pH * rDOS or aeration * initial OD. As the effect of the initial OD was found to be insignificant, the most likely cause is the pH * rDOS two-factor interaction. As such, interactions were considered but all but one was found to be insignificant. However, these might be masked by the strong effect of the pH as already discussed in the manuscript. For all experiments after the initial DoE set we focused on one parameter at a time and therefore disregarded combinatorial effects to keep the number of experiments on a manageable level.

Figure 3-5: Are there any repeated experiments and statistical analysis, otherwise how can the optimization results be explained?

-_-> Statistical analysis was performed for the DoE fermentations, as shown in table 3 and 4. The remaining fermentations were not done in replicates to keep the number of fed-batch fermentations manageable. As such, no statistical analysis could be performed for these fermentations, however the results are in good agreement with the DoE fermentations and therefore are unlikely to be statistical anomalies.

Reviewer 2 Report

Comments and Suggestions for Authors

The authors of this manuscript have established an excellent approach for the large-scale and efficient production of astaxanthin with Corynebacterium glutamicum. The production yield is very impressive, which demonstrates the approach’s great potential for the industrial-scale production of astaxanthin. Overall, the discovery of this study is significant and has the potential to attract researchers in the field. Furthermore, the manuscript is well-written and easy to follow. Therefore, I recommend accepting the manuscript.

 

Author Response

The authors of this manuscript have established an excellent approach for the large-scale and efficient production of astaxanthin with Corynebacterium glutamicum. The production yield is very impressive, which demonstrates the approach’s great potential for the industrial-scale production of astaxanthin. Overall, the discovery of this study is significant and has the potential to attract researchers in the field. Furthermore, the manuscript is well-written and easy to follow. Therefore, I recommend accepting the manuscript.

 

-_-> We thank the reviewer for the recommendation.

Reviewer 3 Report

Comments and Suggestions for Authors

The authors have carried out comprehensive experiments to evaluate various operation factors in fermentation. The results and discussion seem sound and acceptable. Some minor comments:

1. Is the first figure, astaxanthin biosynthesis pathway really needed? 

2. Can the authors discuss why there are optimal parameters pH, rDOS, aeration rate, and inoculation cell density from a physiological aspect?

Author Response

The authors have carried out comprehensive experiments to evaluate various operation factors in fermentation. The results and discussion seem sound and acceptable. Some minor comments:

Is the first figure, astaxanthin biosynthesis pathway really needed? 

-_-> In our opinion it is helpful for the reader to have some context of the previously performed genetic engineering steps in the organism and the possible explanations given in the discussion.

Can the authors discuss why there are optimal parameters pH, rDOS, aeration rate, and inoculation cell density from a physiological aspect?

-_-> All the reactions and corresponding enzymes in an organism possess at least pH optima and their complex interplay leads to there also being optima for growth of the entire organism and production of specific products. For rDOS and aeration rate, the interactions are more difficult to explain, but are likely caused by variations in CO₂ concentrations via various mechanisms as already discussed in the manuscript. For the initial OD we observed no significant effects but it has been an important factor in other fermentations and was therefore analyzed in this work as well.