Improvement of ε-Poly-l-lysine Production by Co-Culture Fermentation Strategy

Round 1

Reviewer 1 Report

In this manuscript, the authors demonstrate that the yield of poly-epsilon-L-lysine can be improved by 13-31% if a co-culture fermentation approach is used as opposed to using a single strain. This result could be interesting to food preservatives industries using this polymer.

There are several areas of improvement before the paper can be accepted for publication:

-e-PL acronym should be defined at the time of its very first occurrence on line 19 rather than later on line 22

Line 35 sounds weird: Rephrase to "containing 25-35 lysine monomer units"

Line 52: cross out "there is"

Line 53: correct to "beneficial" (adjective rather than noun)

Line 54: again sounds very weird. rephrase to "achieved high e-PL accumulation"

Line 60: rephrase to "there had been no" for clarity

Lines 94, 96, 128, 135, 136: Whether centrifuge, vortex, rotary shaker, the g force should also be indicated in addition to rpm value

Materials and Methods: Add a section on Chemicals, describing vendor and analytical grade/purity of all reagents used in the experimental protocols (glucose, ammonium sulphate, ammonia, etc.)

Line 150: What does "suitable multiple" mean?

Line 151: "MeO" ? or rather MeOH?

Figure 1 caption: "Screening of strains" sounds vague and unclear. Be more explicit about what parameters are plotted in the figure!

Line 304: I would say "some other", not "many" because you only provide two examples there (nisin and bioethanol). Also, references should be cited here that evidence what is claimed about these two example substances.

Lines 269, 295, 306, 309 and throughout the manuscript: species binomial names are not always italicized as they should be

Also, references are not presented in MDPI acceptable format. Please adapt accordingly! 

Table 3: I do not see how one can perform an ANOVA when there is only one degree of freedom for so many parameters? It seems to me the sample size is insufficient for this kind of statistical testing.

The English is very poor overall. Urgent professional help should be sought before resubmitting!

Author Response

Please see the attachment.

Author Response File: Author Response.docx

Reviewer 2 Report

This manuscript by Pan et al. dealt with production of ε-poly-L-lysine by co-culture fermentation strategy. The authors used the properly design experiments to meet the objective of the study. Paper possesses good amount of contents and information. With that being said, some work still needed to be polished to improve the overall quality of paper. The authors are strongly suggested to make proper discussion and make data analysis more solid. Furthermore, there are many unbalanced sentences, grammatical errors and typos giving a bumpy flow to the reading. The authors are strongly encouraged to rewrite the whole manuscript for a cohesive and concise presentation of this interesting work. Thus, the reviewer thought this paper need thorough revision before considering for the publication in Fermentation.

Major Comments:

Comment 1: Authors cocultured 5 different strains with S. albulus. Among 5, only C. glutamicum coculture showed the improvement in ε-PL. Authors are suggested to discuss the reason why only C. glutamicum coculture showed improvement and others showed decrement. In addition, authors are suggested to clarify the identification basis for S. albulus and C. glutamicum on the figure.

Authors are also suggested to indicate ratio of co-culture (each strain) inoculation. How much was S. albulus and others strain inoculated?

Comment 2: Author optimized the fermentation parameters like coculture inoculation time, inoculum size, temperature and pH. After coculture inoculation time experiment, it is not clear whether the obtained optimal condition was adapted or not for the subsequent experiment. Authors are suggested to clarify it. Furthermore, authors are suggested to make proper discussion whether production of ε-PL is growth associated or not in regard to the tested parameters.

Comment 3: In the bioreactor experiment, coculture fermentation showed lower cell growth and ε-PL production rate compared to control single culture and there was no much difference in glucose consumption rate. However, the titer of ε-PL increased to 27 g/L. Authors are suggested to discuss the metabolic changes occurred during the fermentation regarding the titer, production rate and yield value. In addition, authors are recommended to discuss the issues of bioreactor and whether flask experiment results were replicated in bioreactor or not.

Comment 4: In the results section, writing is more focused on methods rather than the obtained results. Authors are strongly suggested to analysis the results more and discuss.

Minor Comments:

Comment 1: Authors are suggested to clarify mention the protocol followed.

Comment 2: In methodology section, there is no description about protocol followed for fermentation parameter optimization experiments. Authors are suggested to provide methods for each experiment.

Comment 2: In the 2L fermenter, final volume of media and culture was 1.7L. In addition, there is addition of glucose and ammonia water to maintain the glucose concentration and pH. The rpm was supposed to reach up to 800 rpm. In such high agitation and high volume, there is chances of overflow of fermentation culture. Authors are suggested to clarify it.

Comment 3: In the response surface design method table (Table 2), results were also shown. Authors are suggested to remove results.

Comment 4: Authors are suggested to remove the company name of shaker in Line 168.

Comment 5: Authors are suggested to clarify how much sensitive in temperature probe of shaker. Otherwise, how was temperature (30°C Vs 30.16°C) accurately maintained in shaker.

 

There are many unbalanced sentences, grammatical errors and typos giving a bumpy flow to the reading. The authors are strongly encouraged to rewrite the whole manuscript for a cohesive and concise presentation of this interesting work.

Author Response

Please see the attachment.

Author Response File: Author Response.docx

Round 2

Reviewer 2 Report

Authors responded all the reviewer question properly and modified the manuscript accordingly. The manuscript is acceptable.

Minor english editing is needed.