Review Reports - Prokaryotic Communities from Pristine Cave Environments: Biotechnological Potential with Sustainable Production

Round 1

Reviewer 1 Report

The abstract needs to be rewritten and include the objective of the review, method of how the review was undertaken and the conclusion reached by the author. A review should be more than a compilation of the results reported in literature, it should in fact be a critical assessment of the present knowledge with some clear conclusions what all these results mean, and what are the perspectives and directions for future research and potential applications. So what is the real idea of a review: the reader should afterwards be able to make decisions about what directions to go, what has already been shown to be not promising and what looks promising. It should be a source of inspiration and not just be a collection of facts, it should contribute to the discussion in the field, to be critical and thus result in improving the field. What is needed is to integrate the data and give suggestions for how the gaps can be filled. Kindly revise carefully the manuscript and improve its missing links for further detailed studies.

1. There is no method as to how the review was undertaken.

2. Can be improved by adding the number of studies (hits) pulled by the searches, as well as a thorough description of the study search process.

3. There is no obvious results and discussion but just a flow of information like a book chapter, Need critical and extensive revision

4. The discussion section does not analyze the studies being reviewed - it simply summarizes the findings. Is it the intention of the paper to just put all the information together in one place or is it to critique studies that have already been done?

5. Whole manuscript need extensive revision for further consideration

Author Response

Response to the reviewer 1 comments:

We welcome the valuable and helpful comments that help us to improve our manuscript. The comments were considered, and the manuscript was improved.

Comments and Suggestions for Authors:

R: We had into account the suggestions and the abstract was rewritten, as follows:

“Pristine environments, such as caves, are unique habitats that are isolated from human activity and are exposed to extreme environmental conditions. These environments are rich sources of microbial diversity, and the microorganisms that thrive in these conditions have developed unique survival skills. One such skill is the biosynthesis of secondary metabolites with potential bioactivities, which provide the organisms with a competitive advantage in these extreme environments. The isolation and characterization of microbial strains from the surfaces of pristine cave environments are important for exploring the biotechnological potential of these organisms. These studies can reveal new products with antibacterial, antifungal, anti-inflammatory, antioxidant, and anticancer activities, among others. In addition, the identification of specific compounds responsible for these biological activities can contribute to the development of new drugs and products for sustainable biotechnological applications. Recent developments in genomics, bioinformatics, chemoinformatics, metabolic engineering, and synthetic biology have opened new possibilities for drug discovery, making the exploration of bacterial secondary metabolites more promising. In recent years, several bacteria with bioactive potential have been described and several compounds with bioactivity have been identified. These findings are essential for the development of new drugs and products for the benefit of society. The paper discusses the potential of microorganisms found in pristine cave sur-faces as a source of new metabolites with bioactivity that could have sustainable biotechnological applications. The authors suggest that more research should be conducted in these environments to better understand the microorganisms and the biosynthesis of these metabolites, and to identify new compounds and metabolic pathways that could be of interest for the development of new drugs and products. The aim is to highlight the importance of these habitats as a potential source of new bioactive compounds that could be used for sustainable biotechnological applications.”

There is no method as to how the review was undertaken.
Can be improved by adding the number of studies (hits) pulled by the searches, as well as a thorough description of the study search process.

R: Commentaries were taken into account and this information was included in the methodology section as follows:

“This study aims to consolidate and review the research results of microorganisms in pristine environments, to show the potential that exists in caves and to highlight the importance of further studies in these sites. To facilitate future investigations, we will present the methods currently used to identify microorganisms and their potential activities, as well as the identified bioactive molecules produced by microorganisms from primitive environments. The literature published on the subject throughout the 21st century was thoroughly searched in Scopus to find data for this study. The search terms used were "antimicrobial activity", "anticancer activity", "bioactive compounds", "cave", "microorganisms", and "primitive environments". The search was limited to articles written in English and to years of publication (2000-2023). This allowed the selection of the most interesting articles from the non-specific articles, without omitting those relevant to the review.”

There is no obvious results and discussion but just a flow of information like a book chapter, Need critical and extensive revision.
The discussion section does not analyze the studies being reviewed - it simply summarizes the findings. Is it the intention of the paper to just put all the information together in one place or is it to critique studies that have already been done?
Whole manuscript need extensive revision for further consideration

R: We welcome the suggestions, and the modification was inserted in the manuscript, as follows:

In section 4.1: Identification of microorganisms:

“The identification and study of microorganisms in caves, allows us to understand which species are in the majority and what role they play in the diversity of these environments. Culture-independent methodologies are very useful in this identification, as they allow for a screening of all microorganisms present in a sample. The culture-dependent methods are interesting in the individual study of microorganisms, allowing their isolation for studies of metabolism and the production of compounds with bioactivity.”

In section 4.2: Microorganisms with bioactivity:

“While many studies have been conducted to identify bioactivity in cave bacteria, some of them do not identify the compounds that have activity. This can be due to a variety of reasons, such as the complexity of the microbial community in caves or the limitations of the analytical techniques used. However, identifying the specific compounds that have activity is crucial for further research and development of potential applications. It would be beneficial for future studies to focus on identifying and characterizing the bioactive compounds produced by cave bacteria, as this can lead to a better understanding of their potential uses and applications.”

In section 5.: Conclusions and future directions:

“Advances in techniques for identifying microorganisms and analyzing the compounds they produce have revolutionized our understanding of microbial diversity and metabolism. In the past, many microorganisms and their metabolites went undetected because they were difficult to isolate and culture in the laboratory. However, with the development of new technologies, it is now possible to identify microorganisms and their metabolites without the need for cultivation.

These approaches allow analysis of genetic material, gene expression, and protein function, providing a more comprehensive understanding of microbial diversity and metabolism. As a result, new bacterial species have been discovered and new metabolic pathways that were previously unknown. These discoveries have important implications in fields such as biotechnology, medicine, and environmental science. The discovery of new bacterial strains in caves shows that these environments are important unexplored microbial reservoirs and that their study may give rise to knowledge not only in the area of biotechnology, but also in the discovery of new species, thus increasing phylogenetic understanding and a possible new understanding of the origin of life on Earth, as well as allowing us to better understand the possibilities of life on other planets, because the microorganisms living in these environments are subject to events possibly similar to those on other planets

There are many studies associated with actinobacteria, when compared to the other phyla, in many of the articles reviewed, activity screening was only performed on isolates identified as belonging to the phylum actinobacteria, because they are described as having activities. Studies should be carried out in the remaining phylum, and these should not be discarded, as they have already demonstrated potential anti-microbial and antitumor activities.

Although there are several studies on the bioactivity of microorganisms isolated from caves, there is little information on the chemical structure of the active compounds and the metabolic pathways on which these compounds act and are produced. This situation could be overcome with more frequent use of high-resolution methods such as metagenomics, metatranscriptomics, metabolomics, and metaproteomics which would allow analysis of more information regarding the microorganisms and the biosynthesis of bioactive metabolites produced. Furthermore, by understanding the mechanisms behind the production of compounds with activity by bacteria, it may be possible to increase their production through genetic engineering techniques. This would allow for the sustainable production of these compounds, which could have important applications in fields such as medicine, biotechnology, and agriculture. Genetic engineering techniques can be used to manipulate the genes responsible for the biosynthesis of these compounds, increasing their expression and production. By combining advances in analytical methods with genetic engineering techniques, it may be possible to unlock the full potential of pristine environments and the microbial life that inhabits them.

It would also be interesting to broaden the spectrum of potential bioactivities of cave isolates, allowing the study of anti-inflammatory activity for example, as well as extending the activity of antimicrobials applied to different areas, such as heritage biodegradation.

Continuing research in pristine environments is essential for a better understanding of their ecological functioning and for the discovery of new compounds with potential applications. Bioprospecting and the discovery of new compounds in these environments offer a unique opportunity to study and valorize these natural and cultural heritage habitats while also developing new green, safe, and sustainable solutions. By using low-cost and fast biotechnological processes, it is possible to obtain new products from microorganisms found in these environments. These products can have a wide range of applications, including in the pharmaceutical, agricultural, and environmental industries. They can also be used as more sustainable alternatives to traditional products, reducing environmental impact and promoting a more sustainable approach to industry. Moreover, studying pristine environments can provide important insights into the fundamental processes that underpin ecosystem function, such as nutrient cycling, carbon storage, and microbial interactions. This knowledge can be used to inform conservation strategies and policies to protect these environments, which are often fragile and vulnerable to human activity. Overall, the continued research in pristine environments and the discovery of new compounds offer a promising opportunity for both scientific advancement and sustainable development. By harnessing the potential of these environments and developing new green solutions, we can create a more sustainable and environmentally friendly future.”

Reviewer 2 Report

1- Keywords should be reordered according to the alphabetical

2- Line 58 what dose you mean by the o nutrient-rich

3- Line 73 in my point of view this section is so long the authors wrote extensively and elaborately explaining this part, and it must be merged and shortened so that the reader does not get bored

4- Line 77 with low levels of elements such as ....( this is my opinion )

5- In general If the authors could divide their topics into smaller topics or subtopics, it would somehow make it easier for the reader to follow their research paper

Author Response

Response to the reviewer 2 comments:

We welcome the suggestions, and the alterations were introduced in the manuscript as follow:

Comments and Suggestions for Authors:

Keywords should be reordered according to the alphabetical

R: Commentary was taken into account and the modification was inserted in the manuscript.

Line 58 what dose you mean by the o nutrient-rich

R: To clarify this point, the text was rewritten, as follows:

“The transition zone and the dark zone, are oligotrophic (limited in nutrients), although some may be naturally rich in certain minerals or due to exposure of a nutrient-rich source [16], for example, organic matter present in surface waters or in streams, such as debris, microorganisms, feces, and dead animals [17,18].”

Line 73 in my point of view this section is so long the authors wrote extensively and elaborately explaining this part, and it must be merged and shortened so that the reader does not get bored
In general If the authors could divide their topics into smaller topics or subtopics, it would somehow make it easier for the reader to follow their research paper

R: We had into account the suggestions and the sections were divided into small topics.

Line 77 with low levels of elements such as ....( this is my opinion )

R: Commentary was taken into account and the modification was inserted in the manuscript.

Reviewer 3 Report

The work “Prokaryotic communities from pristine cave environments: biotechnological potential with sustainable production” is promising as we need on sustainable environments. On the other hand, English needs to be improved for a more fluent and understandable reading experience. Furthermore, out-of-context sentences are used to block or interrupt speech at various points throughout the manuscript, making communication difficult at times. You may need to have your manuscript reviewed by a native English speaker or hire a professional English editing service.

This paper aims to systematize is odd phase modify it.

How, salinity, osmolarity, UV radiation, pressure, or pH, with values close to the limit of life. Justify it with proper reference.

I could not find the introduction part in this paper. Include it.

How Pristine environments are linked to microorganisms, pristine environments, antimicrobial activity, anticancer activity. Justify it in the introduction part.

Table 1. Studies of the bioactivity of bacteria from pristine environments are not informative include a column on the molecular mechanism of bioactivity with the illustrative figure.

In section ‘’Potential for bioactive compounds production’’ I could not find any molecular structure of potential for bioactive compounds, include it.

Conclusion and future directions should be included.

Considering all the things, this form of manuscript cannot be published prior to revision. Therefore, I recommended it for major revision.

Author Response

Response to the reviewer 3 comments:

We welcome the suggestions and helpful comments, and the manuscript was rewritten and improved according to the comments as follows:

Comments and Suggestions for Authors

R: We had into account the suggestions and the English has be improved for a more fluent and understandable reading experience.

This paper aims to systematize is odd phase modify it.

How, salinity, osmolarity, UV radiation, pressure, or pH, with values close to the limit of life. Justify it with proper reference.

R: Commentary was taken into account and this information was included in the text:

“Sometimes these environments are exposed to one or more extreme environmental parameters, such as temperature, salinity, osmolarity, UV radiation, pressure, or pH, with values close to the limit of life [2]. These are conditions that make survival im-possible for most life forms. However, there are microorganisms that have adapted their metabolism to live in these environments. These ecosystems can be marine or terrestrial, for example deserts [3,4], arctic sea ice [5,6] and deep sea [4,7,8], where different studies have been conducted to understand how microbial life is possible under these hostile conditions.”

I could not find the introduction part in this paper. Include it. How Pristine environments are linked to microorganisms, pristine environments, antimicrobial activity, anticancer activity. Justify it in the introduction part.

R: The introduction section has been included with your suggestions, as follows:

“Pristine environments can be defined as places with limited or without connections to anthropogenic activities [1]. Sometimes these environments are exposed to one or more extreme environmental parameters, such as temperature, salinity, osmolarity, UV radiation, pressure, or pH, with values close to the limit of life [2]. These are conditions that make survival impossible for most life forms. However, there are microorganisms that have adapted their metabolism to live in these environments. These eco-systems can be marine or terrestrial, for example deserts [3,4], arctic sea ice [5,6] and deep sea [4,7,8], where different studies have been conducted to understand how microbial life is possible under these hostile conditions.

Microorganisms living in unique and extreme environments, such as caves, often develop specialized traits and metabolic pathways due to the selective pressures of their environment. These microorganisms have evolved to survive in nutrient-limited conditions, which often results in the production of unique secondary metabolites. These compounds can have a wide range of bioactivities, including antimicrobial, antifungal, antiviral, and anticancer properties [9], and have the potential to be used in various fields such as agriculture, medicine, and food industry [10,11].

The aim of this review is to provide an overview of the various studies conducted in recent years on the identification of microorganisms in primitive environments and the study of potential bioactive compounds produced by these microorganisms, as well as to review the various methodologies used for these studies.

Our ultimate goal is to study microorganisms isolated from marine, Paleolithic and volcanic caves, with the main objective of finding compounds that have an activity against multi-resistant pathogenic microorganisms that are a public health problem, so that they can be an alternative to antibiotics. We are also analyzing the ability of these compounds to inhibit the proliferation of different tumor cells so that, in the near future, their use as nutraceuticals and/or adjuvants in tumor therapies can be considered. In addition, we are looking at the antioxidant activity of the compounds, which could be beneficial for health in nutrition, pharmacology, cosmetics, or even used in the food sector. In the area of cultural heritage, we are trying to find sustainable alternatives to produce biocides with activity against microorganisms that degrade cultural heritage, in order to apply environmentally friendly products to heritage. Thus, this review al-lows us to have a broad view of all the studies already conducted and to decide which are the best directions to continue the bioprospecting of compounds for sustainable biotechnological application.”

Table 1. Studies of the bioactivity of bacteria from pristine environments are not informative include a column on the molecular mechanism of bioactivity with the illustrative figure.
In section ‘’Potential for bioactive compounds production’’ I could not find any molecular structure of potential for bioactive compounds, include it.

R: Commentary was taken into account and this information was included in legend of table 2.

Conclusion and future directions should be included.

R: We welcome the suggestions, conclusion and future direction was included in manuscript, as follows:

“The caves represent a unique ecosystem in terms of physical, chemical, geological and biological characteristics. The conditions found in these ecosystems are close to the limits of life, so the microorganisms living there have adapted metabolism. This adaptation, which originated thousands of years ago, gives rise to the production of unique secondary metabolites that can be of interest today. Advances in techniques for identifying microorganisms and analyzing the compounds they produce have revolutionized our understanding of microbial diversity and metabolism. In the past, many microorganisms and their metabolites went undetected because they were difficult to isolate and culture in the laboratory. However, with the development of new technologies, it is now possible to identify microorganisms and their metabolites without the need for cultivation.

These approaches allow analysis of genetic material, gene expression, and protein function, providing a more comprehensive understanding of microbial diversity and metabolism. As a result, new bacterial species have been discovered and new metabolic pathways that were previously unknown. These discoveries have important implications in fields such as biotechnology, medicine, and environmental science. The discovery of new bacterial strains in caves shows that these environments are important unexplored microbial reservoirs and that their study may give rise to knowledge not only in the area of biotechnology, but also in the discovery of new species, thus in-creasing phylogenetic understanding and a possible new understanding of the origin of life on Earth, as well as allowing us to better understand the possibilities of life on other planets, because the microorganisms living in these environments are subject to events possibly similar to those on other planets

There are many studies associated with actinobacteria, when compared to the other phyla, in many of the articles reviewed, activity screening was only performed on isolates identified as belonging to the phylum actinobacteria, because they are de-scribed as having activities. Studies should be carried out in the remaining phylum, and these should not be discarded, as they have already demonstrated potential anti-microbial and antitumor activities.

Although there are several studies on the bioactivity of microorganisms isolated from caves, there is little information on the chemical structure of the active com-pounds and the metabolic pathways on which these compounds act and are produced. This situation could be overcome with more frequent use of high-resolution methods such as metagenomics, metatranscriptomics, metabolomics, and metaproteomics which would allow analysis of more information regarding the microorganisms and the biosynthesis of bioactive metabolites produced. Furthermore, by understanding the mechanisms behind the production of compounds with activity by bacteria, it may be possible to increase their production through genetic engineering techniques. This would allow for the sustainable production of these compounds, which could have important applications in fields such as medicine, biotechnology, and agriculture. Genetic engineering techniques can be used to manipulate the genes responsible for the biosynthesis of these compounds, increasing their expression and production. By combining advances in analytical methods with genetic engineering techniques, it may be possible to unlock the full potential of pristine environments and the microbial life that inhabits them.

Round 2

Reviewer 1 Report

Authors have tried to address my concerns. A considerable improvement was observed after revsiion and thus i would recommend the work for publishing.

Reviewer 3 Report

DID THE GREAT WORK