Microbial Diversity Using a Metataxonomic Approach, Associated with Coffee Fermentation Processes in the Department of Quindío, Colombia

Round 1

Reviewer 1 Report

General comments:

- Although the topic is not completely novel, it is a necessary study to contribute to the knowledge of coffee fermentation processes and how it could eventually be implemented to improve coffee production and quality.

- There are still several grammar and redaction errors that make it a bit hard to understand some sentences. Some of them are pointed out in the attached manuscript version with comments. I suggest carefully revising with a native English speaker if it is possible. 

- For specific comments, please check the attached file. 

 

Comments for author File: Comments.pdf

Author Response

Article Ref. FERMENTATION 2311018

Title: Microbial Diversity Using a Metataxonomic Approach, Associated with Coffee Fermentation Processes in the Department of Quindío, Colombia

Authors:  Aida Esther Peñuela-Martínez^1*, Anyela Velasquez-Emiliani ¹ and Carlos A. Angel ^{1, 2}

Affiliations: 1 National Coffee Research Center, Cenicafé., 2 Plant Pathology Independent Scientist

Correspondence*: [email protected] ; Tel.: ((+57) (606) 850 0707 Ext: 4102)

 

RESPONSES TO REVIEWERS:

 

REVIEWER 1

 Line 10: Accepted: Changed relationships by interactions.

• Lines 12- 14: Accepted: Rephased as:  Metataxonomic analysis using high-throughput sequencing and volatile organic compound identification in green coffee beans were performed with HS-SPME and GC–MS.
• Line 16:   Typo AND removed.
• Line 22:   Generate changed by influence.
• Line 75:   Dominant changed by predominant. This suggestion was also included in several paragraphs alog the text.
• Line 80:   Microorganism´s changed by microbial.
• Line 103: Reviewer’s QUESTION: “What was the rationale for selecting only start and ending points? Various studies look for different time points to be able to track the progression of changes within the microbial communities.”  ANSWER:  Most studies in coffee fermentation have been conducted in unique or very limited places, under controlled or specific conditions or steps, and single fermentation types, monitoring sometimes a progressive time course, usually obtaining specific results for those conditions, and limited representativeness of a region. In contrast, our research was conducted in 20 different farms, representative of the Quindío’s coffee agroclimatic zones, in the central region of Colombia, with different coffee production systems, and including several fermentation types as they are performed by the farmers. In addition to conduct microbial diversity, richness and abundance in fermenting coffee samples using High Throughput Sequencing for meta-taxonomy, a widely used strategy, we considered variables for the production system, fermentation parameters, quantified volatile organic compounds in green beans, and established relationships with final coffee beverage quality. However, we added a new paragraph in the Discussion section comparing some specific details in coffee fermentation studies with our results in similar time intervals.  We agree with the Reviewer 1, for future studies, and having additional funding, it would be interesting to have a closer look and detailed analysis for most contrasting locations and fermentation types based on our results. Thank you.
• Line 178: Accepted. Reference included for the used primers Ref. 35: Herlemann, D., Labrenz, M., Jürgens, K. et al. Transitions in bacterial communities along the 2000 km salinity gradient of the Baltic Sea. ISME J 5, 1571–1579 (2011). https://doi.org/10.1038/ismej.2011.41
• Line 180: Accepted. Reference included for the used primers Ref. 36: Tedersoo, L.; Anslan, S.; Bahram, M.; Põlme, S.; Riit, T.; Liiv, I.; Kõljalg, U.; Kisand, V.; Nilsson, R.H.; Hildebrand, F. Shotgun metagenomes and multiple primer pair-barcode combinations of amplicons reveal biases in metabarcoding analyses of fungi. MycoKeys 2015, 1–43.
• Line 182: Accepted: changed purified by trimmed.
• Line 222: Accepted; changed wait by waiting time. Details about fermentation types and process per farm are presented in Table S3.
• Line 230: Accepted: The Figure 2 doesn't really add up any value from what is described in the text.  Instead, Table S3 is now cited in the text, and it have more details.
• Line 235: Accepted: Typo DID, was removed.
• Line 266: Accepted:  Original Figure 3 file is in higher resolution and quality as a .JPEG
• Line 330: Accepted:  Figure 5 color palette was modified to make easier to compare Pseudomonas vs. Frauteria, Leuconostoc vs. Zymomonas, genera Acetobacter vs. Acetobactereaceae...
• Line 375: Accepted: Typo period was removed.
• Lines 402-405:   Sentence rephrased as follows:  By contrast, Cruz-O'Byrne et al. [20], studied fermentations in the northern coffee zone of Colombia, and found that values for these indexes were higher for these, and the richness and diversity for fungi were also greater than for bacterial.
• Line 418: Reviewer QUESTION: “Was there any particular reason to only use SILVA and not integrate with other databases?” ANSWER: based on publications, experience and expert recommendation, SILVA database seem the most complete, updated and appropriately curated to use in this type of general studies.  However, for future studies, we will consider to including additional high-quality databases according to the needs.
• Line 530: Reviewer SUGGESTION: “The discussion points are strong but would have also be interesting to read about how the transition of the communities occurs during the hours of fermentation and maybe comparing to studies that took different (even hourly!) timepoints between start and ending”. ANSWER: A complementary Discussion paragraph with specific details and time frame comparisons were added after Line 468 in the original manuscript submission after Pothakos et al [13]:

These authors found that Enterobacteriaceae (Tatumella, Pectobacterium, Klebsiella, Pantoea, Enterobacter, and Rahnella), and AAB (Acetobacter and Gluconobacter) genera had the highest relative abundance (%) at the beginning of fermentation (0 hours), but it was progressively reduced during the first 8 hours of fermentation, with pH changing from 5.2 to 4.5. In our research, Leuconostoc, Enterobacteriaceae family, Gluconobacter and Tatumella were also with high relative abundance at the beginning (0 hours), with pH values between 5.9 to 4.3. Subsequently, for the second phase from 8-24 hours [13], the most important change was an increase in Leuconostoc and Lactobacillus species, and decrease in some Enterobacteriaceae, with a stable pH of 4.0. Similarly, in most of our studied farms, for traditional fermentation ending from 12 to 22 hours, increase in relative abundance of Leuconostoc and Gluconobacter was observed as well as decrease in Enterobacteriaceae OTUs, with pH between 4.5 and 3.0. By contrast, Cruz-O'Byrne et al. [20], observed a progressive increase Leuconostoc, Acetobacter, Lactobacillus and Weissella, among others in the first 18 to 24 hours of fermentation, with no decrease in the first phase (less than 8 hours).

 

Reviewer 1 General comments:

1- Although the topic is not completely novel, it is a necessary study to contribute to the knowledge of coffee fermentation processes and how it could eventually be implemented to improve coffee production and quality.

ANSWER: In general terms the basic coffee beverage quality is generated in the field, and depending on the harvest, postharvest, processing and beverage preparation, this quality could be maintained or affected (positively or negatively). Our research was focused on exploring the microbial diversity and richness present in coffee farms from several localities and crop conditions, from a very traditional and recognized coffee growing state (Department of Quindío), in the central coffee zone of Colombia. We wanted to know if this diversity was also associated to different fermentation types based on coffee farmers own criteria. We also added to the Conclusion: Despite finding some commonalities in terms of relative abundance of some fungi and bacteria families and genera, there were some specific results or exceptions for individual farms or processes. One of the most important results is that:” There is not a unique or specific method to obtain higher quality coffee or outstanding scores, and the conventional recommended fermentations usually lasting less than 16 hours are still appropriate. There are several myths and some practices that did add much to coffee quality. By contrast, there were some specific practices beyond the recommended ones or problems that happened in steps after traditional fermentation, that could affect coffee quality, generating defects such as fermented, phenol, and earthy.”

 

2)- There are still several grammar and redaction errors that make it a bit hard to understand some sentences. Some of them are pointed out in the attached manuscript version with comments. I suggest carefully revising with a native English speaker if it is possible.

ANSWER: Regarding English spelling, grammar and redaction, the manuscript was sent to MDPI fast edition and revision paid service in advance, and a native English speaker (faculty professor at a land grant University in the Midwest of the U.S.A.) also revised it, and suggested some changes already included in the revised manuscript.

 

We appreciate and thank to the Reviewer 1 for all comments and suggestions to make stronger this manuscript.

 

Reviewer 2 Report

The paper entitled "Microbial Diversity Using a Metataxonomic Approach, Associated with Coffee Fermentation Processes in the Department of Quindío, Colombia" used high-throughput sequencing to assess the microbial composition of spontaneous coffee fermentations. The study is quite comprehensive and provides insight into the differences in microbial community structures across a region of Colombia.

In general, the authors explored a lot of diversity and richness indices and although these are relevant data, I believe that the results of the microbial structure together with the sensory analysis could be better explored because understanding the role and the influence of each microbial group on the sensory profile of coffee is of major importance for the scientific community and especially for the coffee growers. However, the article is well structured and the data generated is relevant to the research area. Regarding the text, here are some suggestions:

Please review if the number corresponding to the NCBI deposited data is correct. Unable to query the archives for this number "PRJNA940080" (accessed 3/23/2023).

Lines 29 - 30: Please review if this information is correct and up to date. There has been a reduction in the 2021/2022 coffee crop in Brazil, the world's largest producer, which may have impacted these numbers. 

Lines 255 -257: I recommend replacing figure 3 with table S2. For example, when we look at figure 3 suggests that 2,3-butanediol is the main compound identified among the alcohols. However, it was only identified in treatment F20 and this happens for other compounds. Also, it would be interesting to make the sensory analysis data available as supplementary material, since it is only mentioned in a general way in the text.

 Lines 257 - 258: It is not possible to make this statement. Some compounds in these chemical classes may indeed be of microbial origin, however, to determine which they are it would be necessary to have performed an analysis of the grains before fermentation.

Figure 6: Please standardize the legend for figures 5, 6, and 8. Remove the "k__", "F__" and "g__".

Author Response

Article Ref. FERMENTATION 2311018

Title: Microbial Diversity Using a Metataxonomic Approach, Associated with Coffee Fermentation Processes in the Department of Quindío, Colombia

Authors:  Aida Esther Peñuela-Martínez^1*, Anyela Velasquez-Emiliani ¹ and Carlos A. Angel ^{1, 2}

Affiliations: 1 National Coffee Research Center, Cenicafé., 2 Plant Pathology Independent Scientist

Correspondence*: [email protected] ; Tel.: ((+57) (606) 850 0707 Ext: 4102)

RESPONSES TO REVIEWERS:

 REVIEWER 2

 SUGGESTION: Please review if the number corresponding to the NCBI deposited data is correct. Unable to query the archives for this number "PRJNA940080" (accessed 3/23/2023).

ANSWER:  The access to the NCBI Bioproject and samples raw data and metadata are already uploaded and were preliminary set to be available in August 2023.  Now, considering that this manuscript could be accepted for publication, access and data availability will be released by A.E. Peñuela-M and Cenicafé as soon as the Fermentation journal editor announces and approves its publication on-line.

 

• Lines 29 - 30: SUGGESTION: Please review if this information is correct and up to date. There has been a reduction in the 2021/2022 coffee crop in Brazil, the world's largest producer, which may have impacted these numbers. ANSWER: Accepted: because it is an interval 2017 -2021/22, the relative production was adjusted between nearly 7 and 9% and ranking was third or fourth, depending on the year.

Despite Brazil had a decrease in production in 202/21 and 2021/22, it is still the largest producer. According to the ICO (International Coffee Organization) from January 2023, report cited and consulted as Reference 1 (https://www.ico.org/documents/cy2022-23/cmr-0123-e.pdf)

“Production and Consumption: The latest provisional estimate for total production in coffee year 2021/22 remains unchanged at 167.2 million bags, a 2.1% decrease as compared to 170.83 million bags in the previous coffee year. World coffee consumption is projected to grow by 3.3% to 170.3 million 60-kg bags in 2021/22 as compared to 164.9 million for coffee year 2020/21. In 2021/22, consumption is estimated to exceed production by 3.1 million bags. The ICO will be publishing shortly new consolidated revised values for production and consumption for 2021/22.”

According to Coffee market Insight at the end of the coffee year 2021/2022 (October 2022) Coffee world production in year 2021/2022 was estimated in 160.4 million, Brazil produced approx. 53.5 M, Vietnam 29.5M, Colombia 11.7M, Indonesia 12.1M. Colombia is still the second largest for Arabica coffee. 2022 Consumption was estimated in 167,5 M, deficit in the market -7.15 M, and 2023 is projected production 171.8 M, surplus in the market +6.4M.

 

• Lines 255 -257: I recommend replacing figure 3 with table S2. For example, when we look at figure 3 suggests that 2,3-butanediol is the main compound identified among the alcohols. However, it was only identified in treatment F20 and this happens for other compounds. Also, it would be interesting to make the sensory analysis data available as supplementary material, since it is only mentioned in a general way in the text.

ANSWER: We respectfully consider it is appropriate to maintain Figure 3 (now Figure 2 in revised manuscript), because it shows a wide spectrum of the whole set of organic volatile compounds present in all samples. In addition, replacing it by Table S2, it is too large to present or change to a main table within the manuscript. We noted the comment of Reviewer 2 and added the sentence citing Table S2 as well: The high concentration of 2,3-butanediol was only found in sample F20 (Table S2).

Regarding to make available the data for sensory analysis as supplementary material, we Accepted and modified Table S1 including two additional columns presenting SCA score (points) average and standard deviation per each farm samples, and the associated defect in coffee beverage, if it was any.

 

• Lines 257 - 258: SUGGESTION: It is not possible to make this statement. Some compounds in these chemical classes may indeed be of microbial origin, however, to determine which they are it would be necessary to have performed an analysis of the grains before fermentation. ANSWER: Accepted, the statement sentences “The first five identified classes were products of microbial metabolism; the remaining three classes were products of thermal reactions in postharvest”, was eliminated, there was not an initial analysis before the fermentation began. In agreement with this observation, a sentence was changed in the revised Abstract (Lines 20-21) as follows: “Alcohols and esters were the main chemical classes identified in green coffee bean samples from these fermentations”, eliminating the part “which were found to be related to microbial metabolism”, from the initial manuscript.

 

• SUGGESTION: Figure 6: Please standardize the legend for Figures 5, 6, and 8. Remove the "k__", "F__" and "g__". ANSWER: Legends were standardized and additional letters were removed.  Figures 3 to 8 were renamed now as Fig. 2 to 7, after eliminating the initial Figure 2, as requested by Reviewer 2.

 

We appreciate and thank to the Reviewer 2 for all comments and suggestions to make stronger this manuscript.

 

Reviewer 3 Report

In this work, the authors studied the potential relationships between some families and genera with different fermentation types and coffee quality.

It is interesting and original. Before the publication, some aspects need minor insights, such as: 

1. Abstract: It is too long (better less than 200 words) and dispersive, the reader couldn’t understand the current needs about the research presented and the final aim and main results. It has to be improved, rewrite it!
2. Introduction: the industrial application of this study isn’t clear. Authors have to deepen the potential industrial applications of their research. First with specific bibliography analysis about the industrial needs of the research section of the work, then describing the final purpose of the work done, in terms of industrial application;
3. Introduction: nothing potential alternative to fermentation of 2nd generation sugars was cited in the paper, an example can consist in the BDO production (De Bari, I., Giuliano, A., Petrone, M.T., Stoppiello, G., Fatta, V., Giardi, C., Razza, F., Novelli, A., 2020. From cardoon lignocellulosic biomass to bio-1,4 butanediol: An integrated biorefinery model. Processes 8, 1–18. https://doi.org/10.3390/pr8121585). Addi t;

4.      Discussion: describe the results obtained and how the current coffee fermentation processes can improve.

 

5. Discussion and conclusions: the research can be interesting, but the application of this study isn’t clear. Authors have to deepen the potential industrial applications of their research, also extending the same analysis to other countries;
6. In general, the authors should describe the advantages of the adopted approach and obtained results also considering the potential environmental/economic improvement.

Author Response

Article Ref. FERMENTATION 2311018

Title: Microbial Diversity Using a Metataxonomic Approach, Associated with Coffee Fermentation Processes in the Department of Quindío, Colombia

Authors:  Aida Esther Peñuela-Martínez^1*, Anyela Velasquez-Emiliani ¹ and Carlos A. Angel ^{1, 2}

Affiliations: 1 National Coffee Research Center, Cenicafé., 2 Plant Pathology Independent Scientist

Correspondence*: [email protected] ; Tel.: ((+57) (606) 850 0707 Ext: 4102)

 

RESPONSES TO REVIEWERS:

REVIEWER 3

It is interesting and original. Before the publication, some aspects need minor insights, such as:

• Reviewer SUGGESTION: Abstract: It is too long (better less than 200 words) and dispersive, the reader couldn’t understand the current needs about the research presented and the final aim and main results. It has to be improved, rewrite it!

ANSWER:  According to Fermentation journal instructions to Authors, the Abstract must be 200 words maximum.  Our manuscript Abstract has 198 words. We understand part of the point from Reviewer 3, however, based on comments from Reviewers 1 and 2, and external comments from MDPI and an English native speaker, faculty professor in related area, we respectfully consider keeping the Abstract content with minor revisions.

To clarify, the current needs about coffee fermentation is to expand the knowledge about microbial communities’ diversity in this process trying to identify microorganisms that could improve the process and quality as stated in the Introduction. In our case, some of the needs are exploring this microbiota in different conditions for crops and processes, the possible role they are playing related to coffee quality by farmers, and its potential to improve beverage quality, added value and differentiation towards higher prices, and special markets or consumers. Our research is considered Exploratory, as many others, also trying to avoid problems and wrong practices during coffee processing that decrease quality. We are taking further steps beyond the previous or current published studies on coffee fermentation, widening the sampled area, number of farms, contrasting environments and crop conditions, and including several fermentation types which were used by regular farmers. There is still large data and results obtained from our analyses, some are work in progress, but we considered publishing the main ones according to the length and scope of the journal, and in agreement with current studies about these topics.

• Reviewer SUGGESTION: Introduction: the industrial application of this study isn’t clear. Authors have to deepen the potential industrial applications of their research. First with specific bibliography analysis about the industrial needs of the research section of the work, then describing the final purpose of the work done, in terms of industrial application.

ANSWER:  We understand the suggestion of Reviewer 2. There is a potential interest from industry to use and formulate products based in microorganisms, enzymes, and metabolites in coffee fermentation, to accelerate it, modify it, or to improve final product quality when used by farmers. Numerous studied have explored this microbiota diversity in this process, and few have focused on specific genera or classes with supported background regarding enzymes and metabolites production. A paragraph was added in the Introduction, Line 48, referring to this topic as suggested by Reviewer 3:

 “Isolation, identification, and characterization of pectinolytic yeasts for starter culture in coffee fermentation has shown the potential to obtain, in this case, pectinase enzymes (pectin lyase, polygalacturonase, and pectin methyl esterase), to accelerate the fermentation process [6], and to improve antioxidants, flavonoids and polyphenols. A previous study using a commercial formulated pectin lyase obtained from Aspergillus niger fungi accelerated degradation of coffee mucilage, with no effects on quality [10]. Native or genetically engineered microbiota are alternatives to improve fermentations, as shown for second-generation sugars to generate energy in biorefineries using non-traditional crops as raw material [11].  A complete recent review on coffee fermentation by Elhalis et al. [5], explores the possible ways to convert the traditional process carried out by microorganisms, into a more controlled and industrialized process, presenting several fungi and bacteria as contributors to fermentation and quality when they are starter cultures or inoculated.

We agree with the Reviewer 3, for future studies, based on our results for contrasting locations and fermentation types, it would be interesting to have a closer look and detailed analysis, and select some of the microorganisms to use them under native or controlled conditions (i.e bioreactors), or produce some metabolites, and their effect of fermentation and coffee quality. Thank you.

• Reviewer SUGGESTION: Introduction: nothing potential alternative to fermentation of 2nd generation sugars was cited in the paper, an example can consist in the BDO production (De Bari, I., Giuliano, A., Petrone, M.T., Stoppiello, G., Fatta, V., Giardi, C., Razza, F., Novelli, A., 2020. From cardoon lignocellulosic biomass to bio-1,4 butanediol: An integrated biorefinery model. Processes 8, 1–18. https://doi.org/10.3390/pr8121585). Add it;

ANSWER:  Accepted: We understand the suggestion of Reviewer 2. As we discussed in previous suggestion and answer, we used as an example the reference provided by Reviewer 2 and added it as Ref 11 to the previous paragraph in the Introduction section:

“Native or genetically engineered microbiota are alternatives to improve fermentations, , as shown for second-generation sugars to generate energy in biorefineries using non-traditional crops as raw material [11]”. In addition, we added to the Conclusions “there is a potential interest from industry to use and formulate products based in microorganisms, enzymes, and metabolites in coffee fermentation, to accelerate or modify it, or to improve final product quality and its added value”.

 

• Reviewer SUGGESTION: Discussion: describe the results obtained and how the current coffee fermentation processes can improve.

ANSWER:  We appreciate Reviewer 3 comment, and respectfully consider that Discussion section already describes the most important results. As we explained before to comments from Reviewer 1 and 2, despite finding some commonalities or differences in terms of relative abundance, diversity, and richness of some fungal and bacteria families and genera, there were some specific results or exceptions for individual farms or processes. For instance, a section of the Discussion already highlighted improvements (Lines 439 to 446 original submitted manuscript) or supported traditional well-conducted fermentations (Lines 461 to 468), whereas another two sections indicated relationships with defects on quality (Lines 468 - 478, and 479 - 493). In addition of two columns in Table S1 have been added to present coffee beverage quality SCA scores and defects data per each farm. We prefer to do not extend more the Discussion section as we have included several paragraphs as requested by Reviewer 3.

 

• Reviewer SUGGESTION: Discussion and conclusions: the research can be interesting, but the application of this study isn’t clear. Authors have to deepen the potential industrial applications of their research, also extending the same analysis to other countries.

ANSWER:  Accepted: a paragraph was added at the beginning of the Discussion: “Our research is relevant, considering that coffee fermentation by the wet method, which is the most traditional way to remove coffee mucilage not only in Colombia, but also in numerous countries, is performed naturally by bacteria and yeasts, mainly. These microbial populations and their interactions along the process influence coffee bean chemical composition and final beverage quality.”.  The following two paragraphs were added to the Conclusions section:  One of the most important results added is that “There is not a unique or specific method to obtain higher quality coffee or outstanding scores, and the conventional recommended fermentations usually lasting less than 16 hours are still appropriate (Table S1)”, and there are several myths and practices that did add much to coffee quality. By contrast, there were some specific practices beyond the recommended ones or problems that happened in steps after traditional fermentation, that could affect coffee quality, generating defects such as fermented, phenol, and earthy.  AND “There is a large potential for farmers and industry to use or formulate products based in native and engineered microorganisms, enzymes, and metabolites in coffee fermentation, to accelerate or modify it, or to improve final product quality and its added value.

 

• In general, the authors should describe the advantages of the adopted approach and obtained results also considering the potential environmental/economic improvement.

ANSWER:  Accepted: We already presented the most important highlights of this type of studies in the Introduction (Lines 49 to 84) and indicated some differences of our research in a relative comparison to previous similar studies in approach and scope (Lines 85 – 98).  As requested by Reviewer 3, some paragraphs have already been added to Discussion and Conclusions sections regarding potential and implications of our research and results.

 

We appreciate and thank to the Reviewer 3 for all comments and suggestions to make stronger this manuscript.