Comparative Analysis of CAZymes from Trichoderma longibrachiatum LMBC 172 Cultured with Three Different Carbon Sources: Sugarcane Bagasse, Tamarind Seeds, and Hemicellulose Simulation

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

Authors are advised to make the following revisions to their manuscript before resubmission for publication in this journal:

1.      Modify the title and content of the manuscript to accurately reflect the study's focus on the medium type (single condition) using three types of media (sugarcane bagasse, tamarind seeds, and hemicellulose simulation).

2.      Shorten the long sentence in the abstract (lines 32-36).

3.      Refrain from using the term "fungus" in the sentence mentioning Trichoderma longibrachiatum and provide more context on why this microorganism is intriguing (Line 76).

4.      Use updated citation regarding Brazil's status as the world's largest sugarcane producer.

5.      Clarify whether the composition mentioned in lines 88-90 refers to sugarcane or sugarcane bagasse.

6.      Discuss the global and/or local generation of sugarcane bagasse, tamarind seeds, and hemicellulose in the introduction section.

7.      Line 93-94: Ensure clarity on whether tamarind seeds are considered an agricultural byproduct, and elucidate their generation process as such.

8.      In the introduction, explain the research gaps related to studies on CAZymes using T. longibrachiatum and the substrates considered in the study to highlight the novelty of the research.

 

9.      With the exception of standalone sections, once the scientific names are written in full in their initial mention, the genus name should be abbreviated in subsequent appearances.

10.  Specify the inoculum size of the fungus.

11.  Please ensure that the medium preparation is clearly described for reproducibility and ease of understanding the experiment. Include details such as the total volume of the medium, concentrations of medium components, and specifically identify the carbon source used. Additionally, clarify why a 1% (w/v) ratio of lignocellulosic residues and hemicellulose simulation was chosen to assess their impact on CAZymes, as this concentration may seem low. It is also unclear from section 2.3 whether sugarcane bagasse, tamarind seeds, and hemicellulose simulation were used individually or as a mixture.

 

12.  Please explain why a control experiment, such as cultivating the fungus without sugarcane bagasse, tamarind seeds, or hemicellulose, was not included.

 

13.   Line 146-147: Did the authors perform centrifugation before filtration? If so, what type of filtration method was employed? Please provide specific details.

 

14.  Number subtopics under the results and discussion section.

15.  Use only essential data for tables; move additional tables to supporting files.

16.  Improve the image quality of Figure 1.

17.  Discuss the specific findings and potential impacts of the study.

18.  Address the limitations and future directions in the conclusions section.

 

 

Comments on the Quality of English Language

Moderate editing of English language required.

Author Response

Reviewer 01:

Authors are advised to make the following revisions to their manuscript before resubmission for publication in this journal:

R.: The authors kindly thank you for your time and revisions. All revisions recommended by the reviewer were performed. We eagerly hope that they meet expectations.

1. Modify the title and content of the manuscript to accurately reflect the study's focus on the medium type (single condition) using three types of media (sugarcane bagasse, tamarind seeds, and hemicellulose simulation).

R.: The title has been modified to accurately reflect the study's focus on the use of three different carbon sources used individually: sugarcane bagasse, tamarind seeds, and hemicellulose simulation (Lines 2-5). Additionally, the content of the manuscript has been updated in several parts to reflect the same idea.

 

2. Shorten the long sentence in the abstract (lines 32-36).

R.: The long sentence was shortened to three shorter sentences (Lines 32-37).

Lines 32-37: Each carbon source showed particularities and differences. Of these, 89 proteins were produced simultaneously with all the carbon sources. Specifically, 41 proteins using only the hemicellulose simulation, 29 proteins when sugarcane bagasse was used as a carbon source, and only 3 when tamarind seeds were used. However, in this last condition, there was a high intensity of xyloglucanase GH74 production, thus reaffirming the richness of xyloglucan in the constitution of these seeds.

 

3. Refrain from using the term "fungus" in the sentence mentioning Trichoderma longibrachiatumand provide more context on why this microorganism is intriguing (Line 76).

R.: The word "fungus" has been removed and more context about why T. longibrachiatum is intriguing has been added (Lines 77-96).

Lines 77-96: One particularly intriguing microorganism is Trichoderma longibrachiatum, especially in the context of biotechnology and biomass bioconversion. T. longibrachiatum is distributed globally, with a predominant presence in warmer climates. Its colonies usually exhibit an initial off-white colony, which later transitions to a shade of greyish green with age [22]. The members of this clade have gained significant attention across different sectors due to their remarkable capacity to excrete substantial quantities of proteins and metabolites [23, 24]. The enzymes produced by T. longibrachiatum are used in various industries, including food, beverages, textiles, and paper, due to their ability to degrade complex plant polysaccharides [25, 26]. Additionally, some strains of Trichoderma, including T. longibrachiatum, are used as biocontrol agents, antagonizing plant pathogens and offering an eco-friendly alternative to chemical pesticides [27, 28]. The study of the CAZymes from the secretome of T. longibrachiatum provides valuable insights into the molecular mechanisms of enzyme production and adaptation to different carbon sources, essential for engineering more efficient strains and understanding their ecological interactions. T. longibrachiatum can grow on a variety of substrates, including agro-industrial residues like sugarcane bagasse and tamarind seeds, making it an ideal model for biomass bioconversion studies and the development of sustainable biotechnological processes [23, 26, 29, 30]. Moreover, the use of T. longibrachiatum in the degradation of agricultural residues not only adds value to these residues but also helps reduce the environmental impact associated with their improper disposal.

 

4. Use updated citation regarding Brazil's status as the world's largest sugarcane producer.

R.: An updated citation regarding Brazil's status as the world's largest sugarcane producer was added.

Reference 33 (Lines 556-557): UNICA. Position on 04/12/2024 [Internet]. 2023/2024 crop ends as the biggest in history. 2018 [Entry 8 July 2024]. Available in: http://www.unica.com.br/

 

5. Clarify whether the composition mentioned in lines 88-90 refers to sugarcane or sugarcane bagasse.

R.: The authors kindly thank you for the observation. The composition mentioned refers to sugarcane bagasse (Line 107).

 

6. Discuss the global and/or local generation of sugarcane bagasse, tamarind seeds, and hemicellulose in the introduction section.

R.: The authors discuss the global generation of sugarcane bagasse (Lines 99-105), tamarind seeds (Lines 112-115), and hemicellulose (Lines 137-139) in the Introduction section.

Sugarcane bagasse (Lines 99-105): Following Brazil, other significant sugarcane-producing countries include India, China, Thailand, and Pakistan. In 2022, global sugarcane production reached a total of 1.92 billion tons, with Brazil producing 38% of the world total, India with 23%, and China producing 5%. These values making it the third most-produced commodity worldwide [34]. Therefore, given that each ton of sugarcane results in approximately 270 kg of bagasse, the global sugarcane crop in 2022 generated more than 500 million tons of bagasse [35].

Tamarind seeds (Lines 112-115): The biggest tamarind producers in the world are countries such as India, Malaysia, Myanmar, Bangladesh, Sri Lanka, Thailand, United Arab Emirates and South American countries. When processing 1 kg of fresh tamarind, it will give 55% pulp, 30-40% seed, 6% peel and 5% fiber [40-42].

Hemicellulose (Lines 137-139): The global annual production of hemicellulose is approximately 60 billion tons, making it the second most abundant renewable component of lignocellulosic biomass, after the cellulose [47].

 

7. Line 93-94: Ensure clarity on whether tamarind seeds are considered an agricultural byproduct and elucidate their generation process as such.

R.: The authors ensure clarity that tamarind seeds are considered a byproduct and elucidate their generation process (Lines 115-124).

Lines 115-124: The seed is the main and underutilized byproduct of the tamarind pulp industry and contains approximately (70%) kernel and (30%) hard brown testa [40-42]. The processing techniques, particularly the removal of pulp from the pod or seeds from the pulp, as well as the handling and storage of the seed and pulp, are traditionally practiced in the growing region or country. However, the most common processing method involves completely removing the testa from the kernel. The testa is separated from the kernels either by roasting or by soaking the seeds in water. Given that the mineral content of the seed coat is higher than that of the cotyledon, it is expected that their thermal properties and behaviors differ, resulting in varying degrees of expansion and contraction. This difference aids in detaching the seed coat from the seed [42].

 

8. In the introduction, explain the research gaps related to studies on CAZymes using T. longibrachiatumand the substrates considered in the study to highlight the novelty of the research.

R.: The authors explain the research gaps related to studies on CAZymes using T. longibrachiatum and the substrates to highlight the novelty of the research in many parts of the Introduction, like Lines 87-91, 105-107, and 115-117. We eagerly hope they suffice for the reviewer.

 

9. With the exception of standalone sections, once the scientific names are written in full in their initial mention, the genus name should be abbreviated in subsequent appearances.

R.: The genus name was abbreviated in subsequent appearances.

 

10. Specify the inoculum size of the fungus.

R.: The inoculum size of the fungus was specified.

Line 164: A spore solution containing 10⁷ spores/mL was prepared from the fungus.

 

11. Please ensure that the medium preparation is clearly described for reproducibility and ease of understanding the experiment. Include details such as the total volume of the medium, concentrations of medium components, and specifically identify the carbon source used. Additionally, clarify why a 1% (w/v) ratio of lignocellulosic residues and hemicellulose simulation was chosen to assess their impact on CAZymes, as this concentration may seem low. It is also unclear from section 2.3 whether sugarcane bagasse, tamarind seeds, and hemicellulose simulation were used individually or as a mixture.

R.: The section 2.3 was rewritten to meet the reviewer's recommendations (Lines 163-180).

Lines 163-180: The submerged culture procedure followed the methodology outlined by Contato et al. [45]. A spore solution containing 10⁷ spores/mL was prepared from the fungus. The fungus was cultured in test tubes, suspended in sterile distilled water, and spore counts were conducted using a microscope and a Neubauer chamber. This suspension was then inoculated into 125 mL Erlenmeyer flasks containing 25 mL of Khanna medium (comprising Khanna's salt solution [20x]: NH₄NO₃ (2.0 g), KH₂PO₄ (1.3 g), MgSO₄.7H₂O (0.362 g), KCl (0.098 g), ZnSO₄.H₂O (0.007 g), MnSO₄.H₂O (0.0138 g), Fe₂(SO₄)₃.6H2O (0.0066 g), CuSO₄.5H₂O (0.0062 g), with distilled water q.s. (100 mL) (5.0 mL); yeast extract (0.1 g); and carbon source (1.0 g); distilled water q.s. to 100 mL) [48]. The media were supplemented, individually, with 1% (w/v) of two different lignocellulosic residues: sugarcane bagasse and tamarind seeds. Additionally, a control simulating hemicellulose was performed, individually, using a mixture containing 0.5% beechwood xylan and 0.5% oat spelt xylan (Sigma-Aldrich, Saint Louis, MO, USA). The 1% (w/v) ratio of lignocellulosic residues and hemicellulose simulation was chosen to evaluate the impact on CAZymes because this concentration was usually reported in studies that successfully evaluated the secretome profile of filamentous fungi [19, 49, 50]. After, the Erlenmeyer flasks were incubated at 30 °C under static conditions for up to 72 h, as optimal conditions for protein induction as described by Contato et al. [45].

 

12. Please explain why a control experiment, such as cultivating the fungus without sugarcane bagasse, tamarind seeds, or hemicellulose, was not included.

R.: The authors thank the observation and clarify that the aim of the work was to use the hemicellulose simulation as a positive control. We have modified the text in several parts to make this intention clear.

We apologize for the absence of a negative control (without sugarcane bagasse, tamarind seeds or hemicellulose), but we clarify that it was not included because the fungus did not develop in the absence of a carbon source, thus making its growth and subsequent analyzes impossible.

 

13. Line 146-147: Did the authors perform centrifugation before filtration? If so, what type of filtration method was employed? Please provide specific details.

R.: The authors did not perform centrifugation before filtration.

The specific details about the filtration were added (Lines 182-184).

Lines 182-184: The culture supernatant of T. longibrachiatum cultivated in the residues under submerged conditions was harvested through filtration with Whatman filter paper Grade 1 in a vacuum pump following a 72-h period.

 

14. Number subtopics under the results and discussion section.

R.: The subtopics under the results and discussion section were numbered.

 

15. Use only essential data for tables; move additional tables to supporting files.

R.: The Tables 5-7 were moved to supporting files.

 

16. Improve the image quality of Figure 1.

R.: The quality of Figure 1 was improved to 300 dpi.

 

17. Discuss the specific findings and potential impacts of the study.

R.: The specific findings and potential impacts of the study were discussed in Lines 392-431.

Lines 392-431: The research identified a total of 206 distinct CAZymes in the secretome of T. longibrachiatum LMBC 172, with 89 proteins consistently produced across all three conditions (sugarcane bagasse, or tamarind seeds, or hemicellulose simulation). Notably, specific proteins were uniquely produced depending on the carbon source, including 41 proteins for hemicellulose simulation, 29 for sugarcane bagasse, and 3 for tamarind seeds. Tamarind seeds, specifically, induced a high production of xyloglucanase GH74, reflecting their high xyloglucan content [44].

The identified CAZymes belong to various families, such as glycosyl hydrolases (GHs), carbohydrate esterases (CEs), polysaccharide lyases (PLs), carbohydrate-binding modules (CBMs), and auxiliary activity enzymes (AAs). The study underscores the adaptive mechanisms of T. longibrachiatum in secreting differential enzymes based on the carbon source, which is crucial for the degradation of specific components of lignocellulosic biomass. This differential enzyme secretion highlights the fungus's ability to utilize diverse carbon sources effectively [23, 24, 26, 29, 66, 67].

From a biotechnological perspective, the detailed secretome analysis of T. longibrachiatum can drive the development of efficient enzyme cocktails for biomass conversion processes, thereby enhancing the production of biofuels and biochemicals [24, 68, 69]. The specific enzymes identified in this study hold potential for further engineering or optimization for various industrial applications, including the food, beverage, textile, and paper industries [25, 68, 70, 71]. Additionally, utilizing agricultural residues like sugarcane bagasse and tamarind seeds as carbon sources for enzyme production promotes sustainable biomass utilization, reducing environmental waste and supporting circular bioeconomy initiatives [14, 26, 45, 72]. The ability of T. longibrachiatum to produce different enzymes tailored to specific substrates suggests potential for customized enzyme production to meet specific industrial needs.

Environmentally, the study supports efforts to mitigate the impact of waste disposal by leveraging agro-industrial residues, thus promoting the efficient use of renewable resources. The findings advocate for eco-friendly alternatives to traditional chemical processing methods, reducing reliance on non-renewable resources and minimizing environmental pollution. Furthermore, this research advances the understanding of molecular mechanisms behind enzyme production and secretion in fungi, contributing significantly to the field of fungal biotechnology. Insights gained from this study can inform future research on other fungal species and their potential applications in various biotechnological processes.

In summary, this study provides a comprehensive analysis of the CAZyme secretome of T. longibrachiatum under different conditions, highlighting significant biotechnological advancements and sustainable industrial applications. The research emphasizes the potential for developing efficient and tailored enzyme solutions to meet the growing demand for sustainable biomass conversion and industrial bioprocessing.

 

18. Address the limitations and future directions in the conclusions section.

R.: The limitations and future directions were addressed in the Conclusions section (Lines 446-451).

Lines 446-451: However, it has limitations that need addressing. The secretome analysis, though detailed, was based on a limited number of carbon sources, and expanding the range of substrates could provide a broader understanding of the enzyme production capabilities of T. longibrachiatum. Future research should explore the genetic and metabolic pathways involved in enzyme regulation and secretion, as well as investigate the synergistic effects of mixed carbon sources on enzyme profiles.

Author Response File: Author Response.pdf

Reviewer 2 Report

Comments and Suggestions for Authors

The author analyzed the enzymes secreted by Trichoderma longibrachiatum cultured under different conditions and identified various families of enzymes, including glycosyl hydrolases (GHs) that cleave cellulose, hemicellulose, and pectin, carbohydrate esterases (CEs), polysaccharide lyases (PLs), carbohydrate-binding modules (CBMs), and auxiliary activity enzymes (AAs). However, there is only a list of results, and no explanation as to why these results occurred. Please discuss why the secreted enzymes differ when using sugarcane bagasse, tamarind seeds, hemicellulose alone, or in combination, based on the composition of the added sugars.

Author Response

Reviewer 02:

The author analyzed the enzymes secreted by Trichoderma longibrachiatum cultured under different conditions and identified various families of enzymes, including glycosyl hydrolases (GHs) that cleave cellulose, hemicellulose, and pectin, carbohydrate esterases (CEs), polysaccharide lyases (PLs), carbohydrate-binding modules (CBMs), and auxiliary activity enzymes (AAs). However, there is only a list of results, and no explanation as to why these results occurred. Please discuss why the secreted enzymes differ when using sugarcane bagasse, tamarind seeds, hemicellulose alone, or in combination, based on the composition of the added sugars.

R.: The authors kindly thank you for your time and revisions. The authors discuss why the secreted enzymes differ when using sugarcane bagasse, or tamarind seeds, or hemicellulose based on the composition of the added sugars (Lines 358-386). We eagerly hope that they meet expectations.

Lines 358-386: The secreted enzymes of T. longibrachiatum differ when using sugarcane bagasse, or tamarind seeds, or hemicellulose simulation due to the distinct composition of the polysaccharides present in these carbon sources. Sugarcane bagasse primarily consists of cellulose, hemicellulose, and lignin, with cellulose being the most abundant polysaccharide [36-38]. As a result, the secretome of T. longibrachiatum grown on sugarcane bagasse is rich in cellulases and other enzymes involved in breaking down cellulose into glucose monomers. Additionally, the presence of hemicellulose and lignin in sugarcane bagasse stimulates the production of hemicellulases (e.g., xylanases) and lignin-degrading auxiliary activity enzymes (AAs), facilitating the comprehensive degradation of this complex biomass.

In contrast, tamarind seeds contain high levels of xyloglucans, which are polysaccharides composed of a cellulose backbone with xylose, galactose, and fucose side chains [43, 44]. The unique structure of xyloglucans in tamarind seeds necessitates the production of specific enzymes, such as xyloglucanases (GH74), to effectively hydrolyze these complex sugars [19]. Consequently, the secretome of T. longibrachiatum cultured with tamarind seeds is particularly enriched in enzymes that target xyloglucans, reflecting the adaptation of the fungus to the predominant polysaccharides in this carbon source.

Hemicellulose simulation, which likely includes a mixture of various hemicellulosic sugars such as xylans, mannans, and arabinogalactans, prompts the secretion of a diverse array of hemicellulases tailored to these components. Enzymes such as xylanases, mannanases, and arabinofuranosidases are produced to degrade the heterogeneous polysaccharide structure of hemicellulose into fermentable sugars [46, 47].

The differences in secreted enzymes when using sugarcane bagasse, or tamarind seeds, or hemicellulose are thus directly influenced by the specific polysaccharide compositions of these substrates. The fungus adapts its enzymatic machinery to efficiently break down the available sugars, producing a tailored set of CAZymes that correspond to the structural complexity and specificities of the given carbon source. This adaptive enzyme production ensures the optimal utilization of the provided biomass, demonstrating the metabolic versatility of T. longibrachiatum.

Author Response File: Author Response.pdf

Round 2

Reviewer 1 Report

Comments and Suggestions for Authors

The authors have adequately addressed my comments and revised the manuscript accordingly. Therefore, the paper can be accepted for publication.

Comments on the Quality of English Language

Minor editing of English language required.

Author Response

Reviewer 01:

The authors have adequately addressed my comments and revised the manuscript accordingly. Therefore, the paper can be accepted for publication.

R.: The authors kindly thank you for your time and revisions. Furthermore, they would like to thank you for accepting the paper for publication.

Author Response File: Author Response.pdf

Reviewer 2 Report

Comments and Suggestions for Authors

The author has responded sincerely to my points and has added appropriate discussion in lines 358-386. I think this section would be better explained with a citation of the Table as the basis for the explanation.

Author Response

Reviewer 02:

The author has responded sincerely to my points and has added appropriate discussion in lines 358-386. I think this section would be better explained with a citation of the Table as the basis for the explanation.

R.: The authors kindly thank you for your time and revisions. The authors used citations from the Tables to better explain. The modifications performed are shown between lines 342-360. We eagerly hope that they meet expectations.

Lines 342-360: As a result, the secretome of T. longibrachiatum grown on sugarcane bagasse is rich in cellulases, such as GH3, GH5, GH6, and GH7, which are crucial for breaking down cellulose into glucose monomers (Tables 1 and 3). Additionally, the presence of hemicellulose and lignin in sugarcane bagasse stimulates the production of hemicellulases (e.g., xylanases, GH10) and lignin-degrading auxiliary activity enzymes (AAs) like AA3 and AA9 (Tables 1 and 3S – Supplementary material), facilitating the comprehensive degradation of this complex biomass.

In contrast, tamarind seeds contain high levels of xyloglucans, which are polysaccharides composed of a cellulose backbone with xylose, galactose, and fucose side chains [43, 44]. The unique structure of xyloglucans in tamarind seeds necessitates the production of specific enzymes, such as xyloglucanases (GH74), to effectively hydrolyze these complex sugars [19]. Consequently, the secretome of T. longibrachiatum cultured with tamarind seeds is particularly enriched in enzymes that target xyloglucans (Table 1), reflecting the adaptation of the fungus to the predominant polysaccharides in this carbon source.

Hemicellulose simulation, which likely includes a mixture of various hemicellulosic sugars such as xylans, mannans, and arabinogalactans, prompts the secretion of a diverse array of hemicellulases tailored to these components. Enzymes such as xylanases (GH11), mannosidases (GH76), and arabinofuranosidases (GH54) are produced to degrade the heterogeneous polysaccharide structure of hemicellulose into fermentable sugars (Tables 1 and 2; and 1S and 2S – Supplementary material) [46, 47].

Author Response File: Author Response.pdf