Rapid Screening of High-Yield Gellan Gum Mutants of Sphingomonas paucimobilis ATCC 31461 by Combining Atmospheric and Room Temperature Plasma Mutation with Near-Infrared Spectroscopy Monitoring

Round 1

Reviewer 1 Report

After completion of the assessment work on this original research manuscript entitled on 'Rapid screening of high-yield gellan gum mutants of sphingomonas paucimobilis ATCC 31461 by combining ARTP mutation with near infrared spectroscopy monitoring', in current form it requires minor improvement in processing temperature in all methodology especially in subsections 2.1 to 2.6.

There are always fluctuation in temperature parameter during processing time. It is not constant at all time. Hence, check the value of temperature parameter mentioned in subsections 2.1 to 2.6. Improvement is required.

Hence, minor revision is essential for further thorough reviewer work. 

Author Response

Response to the reviewer:

Suggestions from the reviewer:

After completion of the assessment work on this original research manuscript entitled on 'Rapid screening of high-yield gellan gum mutants of sphingomonas paucimobilis ATCC 31461 by combining ARTP mutation with near infrared spectroscopy monitoring', in current form it requires minor improvement in processing temperature in all methodology especially in subsections 2.1 to 2.6.

There are always fluctuation in temperature parameter during processing time. It is not constant at all time. Hence, check the value of temperature parameter mentioned in subsections 2.1 to 2.6. Improvement is required.

Hence, minor revision is essential for further thorough reviewer work.

Response: Thank you very much for your kind suggestion. We carefully examined the temperature parameters in the materials and methods especially in subsections 2.1 to 2.6. There are four temperature parameters involved in this study, specifically as follows: 30°C was used for the culture on solid medium or in seed culture medium or in fermentation medium of S. paucimobilis ATCC 31461 strain / mutant strains, 95°C was used for heating fermentation liquid to extract gellan gum, 4°C was used for the precipitation of gellan gum overnight, and 60°C was used for the drying of gellan gum.

Therefore, to avoid confusion, we added the culture temperature of S. paucimobilis ATCC 31461 strain / mutant strains in the section 2.1 as follows: “The S. paucimobilis ATCC 31461 and its mutant strains were cultured at 30 °C in different medium.”, and deleted the description of temperature in subsections 2.1 to 2.6. Thanks again for your nice advice.

Author Response File: Author Response.docx

Reviewer 2 Report

In the present work, Sun et al. describing the ‘Rapid screening of high-yield gellan gum mutants of Sphingomonas paucimobilis ATCC 31461 by combining ARTP mutation with near-infrared spectroscopy monitoring’, I found the work interesting and the data is satisfactory.

Some of the comments are as follows:

1.          Introduction section: authors should add more about the ATRP mutagenesis methods for

 some other microorganisms.  

2.          Page No. 2, line 47-50 (Mutation process of ARTP…………….) suitable reference should be cited.

3.          Manuscript lacking the description of the materials used. The authors should explain the

materials used in all the experiments.

4.          It will be better to start the objective of the manuscript with a new paragraph. (p. no. 2,

line no. 60).

5.          Results: section 3.1, P.no. 4, line 158 reference (Wang et al., 2020) not in a consistent

format.

      6.          Fig. 1A, arrow indication should be described in the figure caption.

      7.         Discussion section seems to be poor. The authors should add some more discussion.

      8.          Some reference is not in a uniform format. The author should correct the same.

   

 

 

Comments for author File: Comments.pdf

Author Response

Response to the reviewer:

Suggestions from the reviewer:

In the present work, Sun et al. describing the ‘Rapid screening of high-yield gellan gum mutants of Sphingomonas paucimobilis ATCC 31461 by combining ARTP mutation with near-infrared spectroscopy monitoring’, I found the work interesting and the data is satisfactory.

Response: Thank you very much for your recommendation. We have tried our best to revise the manuscript according to your kind and constructive comments and suggestions. We sincerely hope that this revised manuscript has addressed all your comments and suggestions.

1. Introduction section: authors should add more about the ATRP mutagenesis methods for some other microorganisms.

Response: Thanks for your constructive suggestion. We have added the introduction about ARTP mutation method for some other microorganisms as follows:

“Xu et al. [10] combined ARTP mutagenesis with 2,4-dinitrophenol (DNP) selection to develop lager yeast, and obtained a serial of strains with higher NADH levels as well as improved flavor stability. Nyabako et al. [11] used ARTP mutation coupled with adaptive laboratory evolution (ALE) to improve the acid tolerance of lactobacillus acidophilus, and obtained the mutant strain LAartp-ale2 with increased lactic acid stress tolerance. Cai et al. [12] reported the improved level of the tyrosine biosynthesis pathway in Saccharomyces cerevisiae through HTZ1 Knockout and ARTP Mutagenesis. Wang et al. [13] combined ARTP mutation with microtiter plate cultivation to screen lycopene-overproducing mu-tants of Blakeslea trispora, and obtained a mutant (WY-239) showing a maximum lyco-pene concentration of 21.80 ± 1.58 mg/g.”

2. Page No. 2, line 47-50 (Mutation process of ARTP…………….) suitable reference should be cited.

Response: Thanks for your kind suggestion. The reference 9 have been cited in this sentence.

 

3. Manuscript lacking the description of the materials used. The authors should explain the materials used in all the experiments.

Response: Thanks for your kind suggestion. We have added the materials “the S. paucimobilis ATCC 31461 or the mutant strain 519” in the methods 2.2-2.8.

4. It will be better to start the objective of the manuscript with a new paragraph. (p. no. 2, line no. 60).

Response: Thanks for your kind suggestion. A new paragraph has been started the objective of the manuscript as suggested.

5. Results: section 3.1, P.no. 4, line 158 reference (Wang et al., 2020) not in a consistent format.

Response: Thanks for your kind suggestion. “Wang et al., 2020” has been deleted and the reference [13] have been inserted, which is in a consistent format.

6. Fig. 1A, arrow indication should be described in the figure caption.

Response: Thanks for your kind suggestion. We have added the figure caption in figure 1A as follows: The arrow in figure 1A indicating the optimal culture time point for ARTP mutation.

7. Discussion section seems to be poor. The authors should add some more discussion.

Response: Thanks for your nice suggestions. We have carefully rewritten the discussion section as follows:

"In recent years, gellan gum has been widely used in food, pharmaceuticals, biomedicine, microbiology, plant tissue culture and many more, due to its excellent gel properties, stability and adaptability, etc [5,6,7]. However, low production yield, high downstream extraction cost and abundant market demand have made gellan gum a high-priced material [26]. Genetic modification is still an important way to increase yield. West [27] used 1% EMS to treat Pseudomonas sp. ATCC 31461 and obtained a mutant strain with elevated production. Arockiasamy et al. [28] analyzed the nonionic surfactants, including Tween 80, Tween 40 and Triton X-100, to improve gellan gum production of S. paucimobilis and obtained the maximum yield (10.44 g/L) with Triton X-100 at 0.75 g/L. Li et al. [7] constructed a carotenoid- and poly-β-hydroxybutyrate-free mutant strain of Sphingomonas elodea ATCC 31461 by knocking out the phytoene desaturase gene (crtI) and phaC gene, and combined UV irradiation and EMS mutagenesis treatment to elevate gellan gum production. However, the similar literatures to elevate gellan gum production were poor. Recently, as a new biological mutagenesis technology, ARTP has been successfully applied in microbial mutagenesis breeding due to its advantages of simple operation, mild conditions, high positive mutagenesis rate and genetic stability [9,11,29]. In this study, the fatality rate of S. paucimobilis ATCC 31461 reached 91.5% after ARTP mutagenesis for 25 s. Based on the high efficiency of mutagenesis when the fatality rate was around 90-95% [13], 25 s was selected as the best ARTP mutation time for the screening high-yield mutant strains. Similarly, Liu et al. [29] showed that the mortality rate of B. coagulans WT-03 reached 93.84% after 15 s of ARTP treatment and selected 15 s as the optimum treatment time to mutate B. coagulans WT-03.

ARTP treatment is often used in combination with other methods to improve mutagenesis efficiency. Liu et al. [29] combined ARTP treatment with ALE to improve the probiotic performance of B. coagulans WT-03. Gu et al. [30] proved that ARTP/EMS-combined mutagenesis efficiently improved production of raw starch-degrading enzymes in Penicillium oxalicum. In the process of ARTP mutagenesis, the high concentration of neutral active ions directly penetrates the cell membrane into the nucleus and causes gene damage, thus causing various types of mutations [9]. Although ARTP has high mutagenesis efficiency, it is a random mutagenesis method. Therefore, only by screening a large number of mutant strains can strains with good performance be screened. In this study, an efficient mutagenesis and rapid screening method of high-yield gellan gum mutants by combining ARTP mutation and NIRS detection technology was proposed for the first time. By using the optimal Normalization pretreatment method and the siPLS regression method, the NIRS model for online predicting the yield of gellan gum in fermentation broth was established, which can replace the traditional alcohol precipitation method to determine the yield of gellan gum. Based on this, the screening efficiency was greatly improved, and the mutant strain library could be expanded to facilitate the screening of more mutant strains with beneficial traits. On the other hand, the alcohol precipitation method, commonly used to determine the yield of gellan gum, requires the use of alcohol or isopropyl alcohol 2-3 times of the fermentation liquid and 1-2 days to determine the yield of gellan gum, which is time-consuming, laborious, and not conducive to environmental protection. The NIRS model established in this study can directly predict the yield of gellan gum in fermentation liquid, saving time, labor and environmental friendly. In addition, the NIRS model can also be used to online predict the yield of gellan gum during fermentation in the industrial production process.

The gellan gum yield of about 600 mutant strains was predicted based on the established ARTP high-efficiency mutation and NIRS model. The yield of gellan gum did not change significantly in most of mutant strains. Significantly, only 17 mutant strains had a higher yield, while about 30 mutant strains had a decreased yield or even almost no gellan gum. It also proved that ARTP mutagenesis is relatively random, and a large number of mutant libraries need to be screened out the strains with high-yield gellan gum. Further, 17 mutant strains were passed for ten generations and gellan gum content was measured by alcohol precipitation method. Only 5 mutant strains had stable gellan gum yield within 10 generations, and the other strains had no significant yield increase. Similar phenomena were also found in the report of Xiang et al. [31].

Among the high yield and stable mutant strains, the mutant strain 519 showed the largest yield improvement. Single-factor and response surface experiments were used to optimize the fermentation conditions of mutant strain 519, and the yield of gellan gum increased by 133.5%. Different fermentation conditions, including carbon sources, nitrogen sources and their concentrations, inoculation amounts, initial pH, culture time in seed solution, and proportion of fermentation liquid to bottle volume, had a great impact on the gellan gum yield of mutant strain 519 ( Figure 5). The most important factors affecting the gellan gum yield of mutant 519 were as follows: soybean meal concentration (A) > inoculation amount (B) > initial pH value (C). In addition, the interaction of soybean meal content, inoculum size and initial pH value had a great influence on gellan gum yield and the interaction between inoculum size and initial pH value was significant (P < 0.05). Therefore, the medium components and their fermentation conditions significantly affect the yield of gellan gum, which was similar to previous reports [32-34]. Zhang et al. [34] improved gellan gum production by optimization of culture medium compositions. Huang et al. [32] revealed that corn steep liquor (CSL), the addition of Triton X-100 surfactant and inorganic nitrogen sources improved the yield of gellan gum. Under the optimal fermentation conditions, the gellan gum yield of mutant strain 519 increased by 133.5%, which laid the foundation for the industrial fermentation of the mutant strain 519. ”

8. Some reference is not in a uniform format. The author should correct the same.

Response: Thanks for your kind suggestion. We have revised the references according the styles of this journal.

Author Response File: Author Response.docx

Reviewer 3 Report

In this research, the time of the treatments has been determined with a very small distance from each other and in the range of seconds. while according to similar studies, a wider time range has been used for the effectiveness of plasma treatment, what is the reference of using the time range? I think choosing treatment in a wider range and longer duration of treatment would have better results.

 

In this research, only the effect of this treatment on mutagenicity was examined,  if possible, it is better to conduct tests, to check whether this mutation causes adverse effects on the samples or not.

 

Line 3: Consider using the complete phrase, instead of "ARTP".

Line 12: It's better to use Complete phrases for the first time, especially in the abstract.

Line 30: It seems that this sentence contains three or more words. Consider to inserting a comma to separate the elements.

Line 32: There is an article problem here, "the acyl" is the correct form.

Line 32: It seems that a verb was missing here. Consider adding it. / are removed

Line 38: It seems there is an article usage problem here. / the industry

Line 43: It is better not to use sentences directly quoted from other studies in the introduction.

Author Response

Response to the reviewer:

Suggestions from the reviewer:

In this research, the time of the treatments has been determined with a very small distance from each other and in the range of seconds. while according to similar studies, a wider time range has been used for the effectiveness of plasma treatment, what is the reference of using the time range? I think choosing treatment in a wider range and longer duration of treatment would have better results.

Response: Thank you very much for your kind suggestion. In the preliminary experiment, we set a wider time gradient of ARTP treatment to analyze the lethality of this bacterium. Based on these results, we set a small distance to obtain accurate treatment time for better results. According to different species treated with ARTP, the treatment time will vary greatly [references 10-13]. The optimal ARTP treatment time is mainly selected when the fatality rate is about 90%. In this paper, when ARTP treatment time was 25s, the fatality rate of this bacterium reached 91.5%. Therefore, 25s was selected as the best ARTP treatment time. Moreover, some mutant strains with high-yield gellan gum were screened in later experiments.

In this research, only the effect of this treatment on mutagenicity was examined,  if possible, it is better to conduct tests, to check whether this mutation causes adverse effects on the samples or not.

Response: Thank you very much for your kind suggestion. Yes, ARTP mutation has both positive and negative effects. In this study, the effect of ARTP treatment on the original strain ATCC 31461 was analyzed with the yield of gellan gum as the evaluation index. As shown in Figure 3, among about 600 mutant strains after ARTP mutagenesis, the yield of gellan gum increased significantly in 17 mutant strains, while that of about 30 mutant strains decreased significantly.

In results 3.3, we have added the sentence as follows:

“Meanwhile, about 30 mutant strains had a significantly decreased yield of gellan gum.”

In discussion, we have added the related discussion as follows:

“The gellan gum yield of about 600 mutant strains was predicted based on the estab-lished ARTP high-efficiency mutation and NIRS model. The yield of gellan gum did not change significantly in most of mutant strains. Significantly, only 17 mutant strains had a higher yield, while about 30 mutant strains had a decreased yield or even almost no gellan gum. It also proved that ARTP mutagenesis is relatively random, and a large number of mutant libraries need to be screened out the strains with high-yield gellan gum.”

Line 3: Consider using the complete phrase, instead of "ARTP".

Response: Thanks for your kind suggestion. "ARTP" was replaced with atmospheric and room temperature plasma.

Line 12: It's better to use Complete phrases for the first time, especially in the abstract.

Response: Thanks for your kind suggestion. The complete phrases “atmospheric and room temperature plasma” was added before "ARTP" in line 12.

Line 30: It seems that this sentence contains three or more words. Consider to inserting a comma to separate the elements.

Response: Thanks for your kind suggestion. We have inserted two commas in the sentence as follows: “The natural gellan gum, directly produced by S. paucimobilis, is high-acyl gellan gum, which is soft and elastic.”

Line 32: There is an article problem here, "the acyl" is the correct form.

Response: Thanks for your kind suggestion. We have revised it in the correct form as follows: “after all or part of the acyl groups”.

Line 32: It seems that a verb was missing here. Consider adding it. / are removed

Response: Thanks for your nice suggestion. We have added it in this sentence as follows: “High-acyl gellan gum form low-acyl gellan gum after all or part of the acyl groups are removed in alkaline conditions”.

Line 38: It seems there is an article usage problem here. / the industry

Response: Thanks for your kind suggestion. We have revised it in the correct form as follows: “in the industry”.

Line 43: It is better not to use sentences directly quoted from other studies in the introduction.

Response: Thanks for your nice suggestion. We have revised these sentences as follows:

“Li et al. [8] combined UV irradiation and ethyl methanesulfonate (EMS) mutagenesis treatment to elevate gellan gum production of a double gene knockout mutant with unexpected production, and obtained a mutant strain with 132.8 % higher than the double gene knockout strain and 14.4 % higher than the wild-type strain ATCC 31461. Li et al. [9] proved that ampicillin, as a stressor and a mutagen, improved exopolysaccharides productivity and viscosity of S. paucimobilis ATCC 31461.”

Author Response File: Author Response.docx

Round 2

Reviewer 3 Report

thanks for your consideration of the comments.