Application of Atmospheric and Room-Temperature Plasma (ARTP) to Microbial Breeding

Round 1

Reviewer 1 Report

The manuscript by Qin Zhang et al. represents a review paper that summarizes a relatively new mutagenesis approach (i.e., ARTP) for the optimization/selection of novel traits of microbes with beneficial properties. The ARTP technology is a relatively novel mutagenesis approach but has recently gained popularity. Such a review is helpful for scientists searching for non-targeted, random-mutagenesis approaches for microbes with poorly developed genetic tools. However, the manuscript should be modified. 

General: 

In its current form, the manuscript represents a list of independent studies, and as such, the review has meager scientific value. The authors should spend more time summarizing the most critical properties of the ARTP, starting from typical set-up, selection strategies, and more comprehensive comparisons with other technologies. It would be great to see a summary of the expected mutation density for different microbial phyla, specific optimization and troubleshooting approaches, etc.  

Comments:

Title/Abstract: ARTP should be defined in the manuscript title and abstract.

Page 2 top: this part needs more specific information, especially in describing mutagenesis approaches and strategies. 

It should be noted that ARTP is a physical method of mutagenesis and should not be placed as a separate methodology. 

Page 3 bottom: ARTP is not just a microbial mutagenesis method and can be applied to other systems (i.e., seeds). Is it wrong? 

SOS repair is a critical element of all biological, physical, and chemical perturbations

Parts 2.1-2.2: This part mostly listed several studies. Some summary/synergy in the approaches should be described. For example, can the authors mention, at least briefly, what mutations enable the success of breeding for Chitosanase production? How many genetic modifications (sites) led to the desired phenotype; what would it require if other methodologies were applied? How was ARTP combined with the clone selection, and what are the most common pathways for selecting desired enzyme producers? 

Part 2.4 Production of PHB should not be confused with environmental remediation of toxic compounds. PHB production could be described as green chemistry/ bio benign plastics, but not remediation. 

Author Response

Comment 2: Title/Abstract: ARTP should be defined in the manuscript title and abstract.

Response 2: This has been done. (See title and Abstract)

Comment 3: Page 2 top: this part needs more specific information, especially in describing mutagenesis approaches and strategies. 

Response 3: This section primarily aims to introduce physical mutagenesis and chemical mutagenesis. Both methods offer various ways to artificially induce mutations. Our focus, however, is not on an in-depth discussion of the mutagenesis techniques themselves, but rather on using a suggestive approach to highlight the advantages of ARTP technology. The emphasis will be on presenting the applications of ARTP.

Comment 4: It should be noted that ARTP is a physical method of mutagenesis and should not be placed as a separate methodology.

Response 4: Your suggestion is very much appreciated. Through literature review, it is evident that ARTP indeed falls under physical mutagenesis. In the article, I have rectified this erroneous description. (See line 72-73)

Comment 5: Page 3 bottom: ARTP is not just a microbial mutagenesis method and can be applied to other systems (i.e., seeds). Is it wrong? 

Response 5: ARTP has a broad range of applications, encompassing microorganisms, plants, and animals. This review mainly focuses on the application of ARTP in the microbial field. However, there are also relevant successful cases of ARTP application in plants.

Comment 6: SOS repair is a critical element of all biological, physical, and chemical perturbations.

Response 6: Regardless of whether it is physical mutagenesis or chemical mutagenesis, after the mutagenic process, the organism will activate its own SOS mechanism for self-repair. During this self-repair process, various mutations such as DNA mispairing and chromosomal inversions can occur, thereby manifesting in the phenotype. Indeed, SOS plays a crucial role as a key element in the mutagenesis process.

Comment 7: Parts 2.1-2.2: This part mostly listed several studies. Some summary/synergy in the approaches should be described. For example, can the authors mention, at least briefly, what mutations enable the success of breeding for Chitosanase production? How many genetic modifications (sites) led to the desired phenotype; what would it require if other methodologies were applied? How was ARTP combined with the clone selection, and what are the most common pathways for selecting desired enzyme producers? 

Response 7：In this review, numerous references have been cited to illustrate the extensive applications of ARTP in the microbial field. However, while each successful case achieved their desired phenotype through ARTP mutagenesis and screening methods, there is still a lack of extensive research concerning detailed metabolic regulation. Undoubtedly, ARTP mutagenesis offers many advantages compared to other mutagenesis techniques. Nevertheless, when focusing on specific traits, different researchers may obtain similar results through various methods, but these outcomes are not generated under identical conditions, rendering them non-comparable. Within this review, several exemplary cases are also presented, elucidating the approaches of integrating ARTP with other mutagenesis methods. (See line 223-236)

Comment 8: Part 2.4 Production of PHB should not be confused with environmental remediation of toxic compounds. PHB production could be described as green chemistry/ bio benign plastics, but not remediation.

Response 8: Indeed, I might have confused the concepts of green chemistry and environmental remediation. In the section concerning environmental remediation, the application of ARTP in this context has already been illustrated through various case studies. (See 285-312)

Reviewer 2 Report

The review article is devoted to the description of the possibilities of a relatively new method for the mutagenesis of microorganisms and algae: Atmospheric and Room Temperature Plasma (ARTP).

Although at first glance the material appears to be quite a lot, it is actually missing a few essential elements.

1. The introduction should be seriously improved by including the history of the method: how it arose as a physical method, who first introduced it in for biological purposes, and the development of the method in historical terms can be considered.

2. The material should be illustrated with at least two or three more tables. The improved mutant bacteria, separately fungi and separately algae, can be described as shown in the table: parent species and strain, target, capabilities, mutant, capabilities, and reference.

3. Figure 2 should be edited by correcting any grammatical errors in it, for example, Speed ability, Application ... to microorganisms, Biological enzymes, and Function. In addition, the authors should avoid the term "biological enzymes"; "enzymes" is the appropriate term. "Breeding" cannot be used for microorganisms, the authors should correct it thorough the text.

4. ARTP must be written in full in both the title and the abstract. In the third paragraph on page 2, the method is written differently.

5. References in the text are not, as required in journals in the MDPI group, to be placed in square brackets with numbers in order of mention in the text. Correct the citations in the reference list.

6. Titles of subsections to be edited, for example, 2.1. it is better to make "Application to Enzyme Overproducers" similar to section 3.

7. "Outlook" would sound better as "Future Perspectives".

The article is a product of machine translation and needs polishing, stylistic corrections, as well as any spelling errors.

Author Response

Comment 1: 1. The introduction should be seriously improved by including the history of the method: how it arose as a physical method, who first introduced it in for biological purposes, and the development of the method in historical terms can be considered.

Response 1: In the article, we have incorporated additional information about the historical development and functionalities of the ARTP mutagenesis device. Some review articles have already covered the historical development of ARTP. Furthermore, the introduction elaborates on various factors and key elements contributing to the successful implementation of ARTP mutagenesis. (See Line 74-79, Line 24-149)

Comment 2: The material should be illustrated with at least two or three more tables. The improved mutant bacteria, separately fungi and separately algae, can be described as shown in the table: parent species and strain, target, capabilities, mutant, capabilities, and reference.

Response 2: A detailed explanation was provided using three tables. The tables comprehensively depict the applications of ARTP in microorganisms over the past 5 years, the method, compound, ability and the variations in ARTP mutagenesis time among different strains.

Comment 3: Figure 2 should be edited by correcting any grammatical errors in it, for example, Speed ability, Application ... to microorganisms, Biological enzymes, and Function. In addition, the authors should avoid the term "biological enzymes"; "enzymes" is the appropriate term. "Breeding" cannot be used for microorganisms, the authors should correct it thorough the text.

Response 3: Figure 2 has been re-edited. Through literature review, some articles also utilized the term "Microbial breeding."

Comment 4: ARTP must be written in full in both the title and the abstract. In the third paragraph on page 2, the method is written differently.

Response 4:  Have done.

Comment 5: References in the text are not, as required in journals in the MDPI group, to be placed in square brackets with numbers in order of mention in the text. Correct the citations in the reference list.

Response 5: Have done

Comment 6: Titles of subsections to be edited, for example, 2.1. it is better to make "Application to Enzyme Overproducers" similar to section 3.

Response 6: Have done.

Comment 7: "Outlook" would sound better as "Future Perspectives".

Response 7: Have done.

Comment 15: Comments on the Quality of English Language

The article is a product of machine translation and needs polishing, stylistic corrections, as well as any spelling errors.

Response 15: We have submitted the article to a professional English editing service for proofreading and editing.

Round 2

Reviewer 1 Report

The manuscript was sufficiently improved. I do not have additional suggestions.

Reviewer 2 Report

The manuscript was improved according to the requirements. It is suitable for publication now.