Integrated Metagenomics and Network Analysis of Metabolic Functional Genes in the Microbial Community of Chinese Fermentation Pits

Round 1

Reviewer 1 Report

This manuscript is about the analysis of metabolic functional genes for the microbes in fermentation pits. The author have done some experiments and obtained a lot of data. However, some issues were not addressed clearly and it can provide little new insight in this aspect.

Other comments:

1. The authors didn’t explain why the samples are “representative” and from which stage of fermentation the samples were taken.

2. It seems that the authors failed to find significant difference in metabolic paths among the pits of different ages. Most of the common metabolic pathways may not of special meaning for liquor brewing.

3. The authors didn’t indicate the potential contribution of different pits. Generally speaking, the production of flavor compounds are only partly related with the microorganisms from pits.

Author Response

Response to Reviewer 1 Comments

 

Point 1: The authors didn’t explain why the samples are “representative” and from which stage of fermentation the samples were taken.

 

Response 1: Firstly, thank you very much for your valuable and helpful comments concerning our manuscript. In order to ensure representativeness, the samples were collected from the most representative famous traditional company in China. The microorganisms are dynamically changing during the fermentation process. So the sampling was carried out at the end of each fermentation cycle after emptying the fermented cereals. The related content has been complementally described in revised manuscript.

 

Point 2: It seems that the authors failed to find significant difference in metabolic paths among the pits of different ages. Most of the common metabolic pathways may not of special meaning for liquor brewing.

 

Response 2: It’s obvious that the metabolic pathways have significant differences from other environments, such as the compared typical agricultural soil in the manuscript. However, the domestication process occurred after the long period of CSAB fermentation, which may lead to the convergence of microbial metabolic pathways. Another possibility is that the genes can be annotated based on the existing public databases. Moreover, a substantial proportion of unknown functional genes accounting for 23.78% had been detected. The significant differences may mainly lie within these unknown genes, but current research indicates that they cannot be effectively deciphered. There is still a limited understanding of these genes associated with the flavor compounds. It still needs to be deeply studied in the following work as the improvement of research level.

 

Point 3: The authors didn’t indicate the potential contribution of different pits. Generally speaking, the production of flavor compounds are only partly related with the microorganisms from pits.

 

Response 3: Indeed, as the described in the manuscript that the differences in the functional gene distribution of microbial ecosystems in FPs were not significant; however, the characteristics were only suitable for the requirement of metabolic flavor compounds in the fermentation production of CSAB. However, subtle differences were observed in key metabolic pathways. These subtle adjustments in the metabolism of the abovementioned substance categories led to differences in CSAB quality. For instance, as shown in Fig. 5, the functional categories of [C] (energy production and conversion), [E] (amino acid transport and metabolism), [G] (carbohydrate transport and metabolism), and [I] (lipid transport and metabolism) increased in abundance with an increase in age. Therefore, this work has been confirmed that the quantitative distribution and the functional category of genes have certain distinction in different FPs. The production of flavor compounds is affected by many factors, such as microorganisms, fermentative parameters, raw materials, and environmental conditions. Indeed, only one factor could not completely determine the production of flavor compounds.

 

Special thanks to you for your helpful guides.

Reviewer 2 Report

The work is devoted to the study of the molecular bases that allow the microbiome of fermentation pits (BB) to carry out the fermentation of Chinese strong-aromatic baijiu (CSAB). This work is a continuation of the work “Characterization of microbial community profiles associated with quality of Chinese strong-aromatic liquor through metagenomics” https://doi.org/10.1111/jam.14279 , in which  i) samples were taken (representative famous FPs of CSAL, in Luzhou city (28.8833°N, 105.4500°E), Chengdu city (30.6586°N, 104.0647°E) and Mianyang city (31.4667°N, 104.6833°E), China), ii) isolated metagenomic DNA and iii) performed metagenomic sequencing, obtaining total of ~3_4 billion sequence reads, including nearly 337 Gb (gigabases) of metagenomic sequence data, iv) carried out genotyping of bacterial microbiomes by 16/18S rRNA sequences.

In the current study, the authors performed an additional bioinformative analysis (related to characterization of microbial genes) of previously obtained raw data on whole genome sequencing of FPs metagenomes. In this regard, the Materials and Methods section should not repeat (word by word) previously published methods in the section 2.1 Sampling and metagenomic DNA Extraction and  2.2 Paired-end (PE) library generation and Illumina Hiseq sequencing. It is necessary to refer to the previous work, as a result of which the current data were obtained, on the basis of which the bioinformatic analysis was carried out.

In addition, it is necessary for the authors to make the raw data described in the article available to the scientific community by posting them online.

In previous work, when the authors took samples of microorganisms (then isolated DNA from them, carried out whole genome sequencing of metagenomic DNA and obtained raw data on these sequences), they called the drink Chinese strong-aromatic-liquor (CSAL). Now the same samples are associated with the preparation of the Chinese strong-aromatic baijiu (CSAB). To avoid confusion for readers, the current article should say in the Introduction that CSAB is the previously described CSAL.

The work carried out a significant bioinformatic analysis, characterized several thousand genes in the composition of the microbiome, determined their possible role in the production CSAB. In general, the work was done at a good methodological level, most of the results are clearly presented, the totality of the data obtained allows us to understand some patterns in the fermentation of CSAB in the studied regions of China at the metabolic level.

There are several remarks after which the work can be published.

 

Comments:

Lines 2-4

Reword the title of the article to make it clearer, in particular avoiding generic terms such as "diverse characteristics".

 

Lines 17-20

 

You say you've found 1,375,660 microbial genes. Clearly, there are many repeats among the 1,375,660 microbial genes. You say you've found 3,379 ubiquitously known gene clusters. But also indicate how many unique sequences of known genes (not clusters) turned out to be among 1,375,660 genes and how many unique genes (both known and not known) were among 1,375,660 decoded sequences.

  

Lines 24-25

 

In a previous work https://doi.org/10.1111/jam.14279 , you characterized the fermentation pits (FPs) microbiome only in terms of bacteria (with the exception of Figure 1a, where you give information about the total composition of eukaryotes). In the current article, give full information about the studied microbiomes, supplementing with data on fungi. This is important to understand the contribution of bacteria and fungi to the fermentation process being studied. Moreover, you speak in a number of cases about the significance of the metabolism of yeast fungi), for example, you say yeast-type alcoholic fermentation metabolic pathway of Saccharomycetales (Line 469-470).

  

Line 225 (Results and Discussion)

 

The microbiome of fermentation pits (FPs) is studied in this work; however, in different regions of China, it obviously has a different structure. In this regard, add sampling locations on the geographic map of China. Without such a sampling map, it is not clear to a foreign reader which regions of China are covered. With a map, it will become clear that all the samples were taken in a relatively close area of ​​central China. Justify why this particular region of central China was chosen for the study, why it is interesting from a historical and practical point of view for obtaining CSAB.

 

Figure 5. In its current form, this figure is not readable (highly compressed) and is for informational purposes only, most of the textual information on it is lost. Ask the MDPI publishers (at the stage of preparing the article for publication, when the article is accepted) to add a higher resolution layout of the article Figure 5 to the pdf.

Author Response

Response to Reviewer 2 Comments

 

The work is devoted to the study of the molecular bases that allow the microbiome of fermentation pits (BB) to carry out the fermentation of Chinese strong-aromatic baijiu (CSAB). This work is a continuation of the work “Characterization of microbial community profiles associated with quality of Chinese strong-aromatic liquor through metagenomics” https://doi.org/10.1111/jam.14279 , in which  i) samples were taken (representative famous FPs of CSAL, in Luzhou city (28.8833°N, 105.4500°E), Chengdu city (30.6586°N, 104.0647°E) and Mianyang city (31.4667°N, 104.6833°E), China), ii) isolated metagenomic DNA and iii) performed metagenomic sequencing, obtaining total of ~3_4 billion sequence reads, including nearly 337 Gb (gigabases) of metagenomic sequence data, iv) carried out genotyping of bacterial microbiomes by 16/18S rRNA sequences.

 

In the current study, the authors performed an additional bioinformative analysis (related to characterization of microbial genes) of previously obtained raw data on whole genome sequencing of FPs metagenomes. In this regard, the Materials and Methods section should not repeat (word by word) previously published methods in the section 2.1 Sampling and metagenomic DNA Extraction and 2.2 Paired-end (PE) library generation and Illumina Hiseq sequencing. It is necessary to refer to the previous work, as a result of which the current data were obtained, on the basis of which the bioinformatic analysis was carried out.

 

Response 1: Firstly, thank you very much for your comments and in-depth understanding of our work. And we are grateful to the reviewer for the valuable suggestions. The previous work has been referred in the revised manuscript. In addition, the main duplicate content has been deleted and adjusted.

 

In addition, it is necessary for the authors to make the raw data described in the article available to the scientific community by posting them online.

 

Response 2: The raw data of metagenomes have been deposited at the NCBI Sequence Read Archive under the accession numbers SRX691263 to SRX691265 and SRX691272 to SRX691280. It had been reported in the authors' previous work https://doi.org/10.1128/genomeA.01045-14.

 

In previous work, when the authors took samples of microorganisms (then isolated DNA from them, carried out whole genome sequencing of metagenomic DNA and obtained raw data on these sequences), they called the drink Chinese strong-aromatic-liquor (CSAL). Now the same samples are associated with the preparation of the Chinese strong-aromatic baijiu (CSAB). To avoid confusion for readers, the current article should say in the Introduction that CSAB is the previously described CSAL.

 

Response 3: The China National Standardization Management Committee had implemented a standard: GB/T 17204-2021 “Terminology and classification of alcoholic beverages” on 1 June 2022. The word “baijiu” becomes the standard name. Therefore, we changed “liquor” to “baijiu”. The alias of CSAB has been supplementarily described in the Introduction according to the reviewer’s suggestions.

 

The work carried out a significant bioinformatic analysis, characterized several thousand genes in the composition of the microbiome, determined their possible role in the production CSAB. In general, the work was done at a good methodological level, most of the results are clearly presented, the totality of the data obtained allows us to understand some patterns in the fermentation of CSAB in the studied regions of China at the metabolic level.

 

There are several remarks after which the work can be published.

 

Response 4: Thanks for your appreciation of the work.

 

Point 5: Lines 2-4

Reword the title of the article to make it clearer, in particular avoiding generic terms such as "diverse characteristics".

 

Response 5: The meaningless words have been deleted according to the Reviewer’s suggestion.

 

Point 6: Lines 17-20

You say you've found 1,375,660 microbial genes. Clearly, there are many repeats among the 1,375,660 microbial genes. You say you've found 3,379 ubiquitously known gene clusters. But also indicate how many unique sequences of known genes (not clusters) turned out to be among 1,375,660 genes and how many unique genes (both known and not known) were among 1,375,660 decoded sequences.

 

Response 6: The predicted genes were subjected to redundancy removal using the CD-HIT tool. The 1,375,660 genes were the total of non-redundant genes predicted from all the FPs. However, the 3,379 ubiquitously genes were the co-contained known functional genes among the different ages, layers, and geographical regions of FPs deciphered by Venn analysis. Therefore, we defined these 3,379 genes as the “core functional gene catalog” of FPs. These two number respectively represent different meanings. The relevant statements were in the line 269-275 and 333-336 of the revised manuscript.

 

Point 7: Lines 24-25

In a previous work https://doi.org/10.1111/jam.14279 , you characterized the fermentation pits (FPs) microbiome only in terms of bacteria (with the exception of Figure 1a, where you give information about the total composition of eukaryotes). In the current article, give full information about the studied microbiomes, supplementing with data on fungi. This is important to understand the contribution of bacteria and fungi to the fermentation process being studied. Moreover, you speak in a number of cases about the significance of the metabolism of yeast fungi), for example, you say yeast-type alcoholic fermentation metabolic pathway of Saccharomycetales (Line 469-470).

 

Response 7: We thank the reviewer very much for this helpful suggestions. The detailed information about the bacteria and fungi in the FPs has been given by Supporting Table S1 and S2 in the Supplementary File. And the related supporting tables had been referred in Line 466 and 476.

 

Point 8: Line 225 (Results and Discussion)

The microbiome of fermentation pits (FPs) is studied in this work; however, in different regions of China, it obviously has a different structure. In this regard, add sampling locations on the geographic map of China. Without such a sampling map, it is not clear to a foreign reader which regions of China are covered. With a map, it will become clear that all the samples were taken in a relatively close area of central China. Justify why this particular region of central China was chosen for the study, why it is interesting from a historical and practical point of view for obtaining CSAB.

 

Response 8: This is an excellent suggestion. In response, a sampling map has been added as Figure 1 in the revised manuscript. Indeed, it's beneficial to clearly understand the sampling information of CSAB.

 

Point 9: Figure 5. In its current form, this figure is not readable (highly compressed) and is for informational purposes only, most of the textual information on it is lost. Ask the MDPI publishers (at the stage of preparing the article for publication, when the article is accepted) to add a higher resolution layout of the article Figure 5 to the pdf.

 

Response 9: Thanks to the Reviewer’s meticulous consideration. We wolud like to provide the Figure 5 in the editable format of Abobe Illustratоr by the email attachments.

 

Special thanks to you for your helpful guides.

Round 2

Reviewer 1 Report

The revisions are acceptable

Reviewer 2 Report

The authors took into account all the comments, added a figure with a map of sampling in China. This made the manuscript more coherent. In its current form, it can be accepted for publication