Development of a Molasses-Based Medium for Agrobacterium tumefaciens Fermentation for Application in Plant-Based Recombinant Protein Production

Round 1

Reviewer 1 Report

The aim of the manuscript was the development of a molasses-based medium of Agrobacterium tumefaciens fermentation as a tool for plant-based recombinant
protein production.

Although it is an interesting work, there are a few issues of major importance that should be addressed prior final consideration for publication.

p. 4. “Meanwhile, the highest cell growth was observed in the Initial basal medium, where the OD600 reached 1.9 after 24 h incubation, and this growth was comparable with the growth of A. tumefaciens EHA105-pEAQ in LB medium. Interestingly, use of either Sucrose-based defined media or Initial basal medium for A. tumefaciens GV3101-pEAQ cultivation resulted in high cell growth with sucrose as a sole C-source. The OD600 at 24 h incubation in both media were compatible with LB medium growth where the OD600 reached 2.0 (Figure 1b).”. Please also provide cell counts, not only absorbance values.

p. 6. “However, A. tumefaciens GV3101-pEAQ preferred the Sucrose-based defined medium for growth than SA-Molasses based medium (Figure 2b). Meanwhile, TC and TCSA- Molasses based medium showed a similar result. The growth of A. tumefaciens EHA105-pEAQ in TC and TCSA- Molasses based medium was as good as the growth in Sucrose-based defined medium (Figure 2a), but significantly lower growth rate of both Molasses containing medium was observed in A. tumefaciens GV3101-pEAQ (Figure 2b)”. Any possible explanation?

p. 7. “The supplementation with Fe2+ slightly affected the growth of A. tumefaciens EHA105-pEAQ but not with the calcium and manganease supplementation (Figure 2a). In contrast, supplementation with Fe2+ Mn2+ and Zn2+ in Molasses based medium, resulted in growth inhibition of A. tumefaciens GV3101-pEAQ (Figure 2b)”. Any possible explanation?

Discussion. This section needs substantial improvement. The authors should discuss and compare their findings with results previously reported in the literature rather than presenting again their data.

Conclusions are missing.

Author Response

Please see the attachment.

Author Response File: Author Response.pdf

Reviewer 2 Report

Comments to Authors

-The development of a molasses medium is interesting and follow the recent research trend: However, please check the underlisted suggestions:

TITLE:

-You may consider using another word "as a tool" seems not suitable.

"as alternative stimulant"

ABSTRACT:

-include some statistical facts or results in your abstract.

INTRODUCTION:

-The last paragraph of the introduction: the part sound like conclusion. I suggest you improve on the introduction of the manuscript. It looks too small and no detail and lack recent and updated information as related to your current study.

-End the latter part of this introduction with the clear aim of the study.

MATERIALS AND METHODS:

-section 2.1: How did you get the strain? Was it purchase or receive as a gift or isolated newly? that information is missing.

-Agitation was done where?

Section 2.2:

-Section this way: 2.2.1 subsection for SA

2.2.2 subsection for TC

2.2.3 subsection for TCSA

-what is the model of the centrifuge?

Section 2.3:

--Reconstruct table 1 to reflect only the different compositions in the mentioned medium composition and mentioned the similar ones as part of section 2.3

Section 2.7: The economic evaluation is not done based on just cost evaluation?

RESULTS

--Section 3:1: Provide literature evidence to support your results in section 3.1, your current discussion is more of observations

-Section 3.2:

--"This is because the untreated cane molasses contained high concentrations of metal ions, suspended colloids and ash, that were inevitably generated during cane molasses refining that result in cell growth inhibition."

Ascertain this claim please, you either show results or provide suitable 

Section 3.5:

-The economic evaluation is just a price comparison without basis? So you have not done any economic evaluation as far as i know?

-What is the basis for the economic evaluation?

-What is the price index for the medium composition?

-Price basis year?

DISCUSSION

I suggest you combine your results and discussion. Discuss each of the results section in detail.

There will be need to include conclusion if you consider combining results and discussions.

References:

-Add recent and updated references

Please: You can also check the attached reviewed pdf for other minor corrections directly.

Comments for author File: Comments.pdf

Author Response

Please see the attachment.

Author Response File: Author Response.pdf

Round 2

Reviewer 1 Report

The paper has been significantly improved and I think it is now suitable for publication.

Reviewer 2 Report

The authors have addressed the comments.