Role of In-House Enzymatic Cocktails from Endophytic Fungi in the Saccharification of Corn Wastes Towards a Sustainable and Integrated Biorefinery Approach

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

The authors of the manuscript fermentation-3412046 investigated the  biosynthesis of a enzymatic cocktail (cellulases and hemicellulases) by  fungal strain of  Beauveria bassiana, Trichoderma asperellum, Metarhizium anisopliae and Pochonia chlamydospore. The biosynthesis was done in solid state fermentation (SSF), using sugarcane bagasse (SB) and wheat bran (WB) for the growth of the tested fungi.

The main manuscript weakness is related to statistical analysis. Authors made experiments in triplicate.  Figures 1 and 2 mentioned “Values represent mean values of experiments performed in triplicate (p < 0.05).” However, some of the data from these figures are presented with error bars. Other data do not have error bars. What represent these bars (standard deviation, standard error) is not mentioned. In Figure 3 caption author mentioned “ The results are expressed as average ± standard (sic!) deviation (n = 3).” The data are represented without standard deviation.

The authors must complete Figure 1 and Figure 2 captions with the information related to error bars.  The data from the Figures must be corrected, with error bars on each  point. The info from L158-L160 must be completed (e.g., used software). Statistical analysis was  performed also for other  experiments, not only for determination of enzymatic activities. Therefore, the statistical analysis must be described as a separate sub-section.

Strains designation  and scientific proof of strains identifications must be  presented in section 2.1. Microorganisms. A statement of the availability of these strains from the y H.T.M. Comercio e Laboratórios de Corretivos do Solo Ltda - Biosag 110 (Ituverava, SP, Brazil) must be included.  Without these info, the reproducibility of the presented data (by other scientists) are questionable.

Manuscript need to be carefully  rewritten, to improve precision and clarity. For example, L259-260, “Concerning M. anisopliae, this fungus has been well explored as biopesticide agent for plant defense…” The correct word that must be used is “protection” and not “defense”.

Author Response

Reviewer 1

The authors of the manuscript fermentation-3412046 investigated the  biosynthesis of a enzymatic cocktail (cellulases and hemicellulases) by  fungal strain of  Beauveria bassiana, Trichoderma asperellum, Metarhizium anisopliae and Pochonia chlamydospore. The biosynthesis was done in solid state fermentation (SSF), using sugarcane bagasse (SB) and wheat bran (WB) for the growth of the tested fungi.

1. The main manuscript weakness is related to statistical analysis. Authors made experiments in triplicate.  Figures 1 and 2 mentioned “Values represent mean values of experiments performed in triplicate (p < 0.05).” However, some of the data from these figures are presented with error bars. Other data do not have error bars. What represent these bars (standard deviation, standard error) is not mentioned. In Figure 3 caption author mentioned “ The results are expressed as average ± standard (sic!) deviation (n = 3).” The data are represented without standard deviation.

Response:

Figures 1,2 and 3 error bars: We have updated the captions for Figures 1 and 2 to include detailed information about the error bars, specifying that they represent the standard deviation of the mean values. Additionally, all data points in the figures now include error bars, as requested.

Figure 3 caption: As specified in the caption of Figure 3, error bars representing the standard deviation (n = 3) have been added to each data point in the graph.

Tables 1 and 2: All numerical results are now presented with their respective standard deviations.

 

2. The authors must complete Figure 1 and Figure 2 captions with the information related to error bars.  The data from the Figures must be corrected, with error bars on each  point. The info from L158-L160 must be completed (e.g., used software). Statistical analysis was  performed also for other  experiments, not only for determination of enzymatic activities. Therefore, the statistical analysis must be described as a separate sub-section.

Response:

The text in this section has been completed with information about the software used for statistical analysis, as requested (L238 –L242).

Statistical analysis description: A new subsection titled “2.7 Data Analysis” has been added to the Materials and Methods section, providing description of the statistical methods applied the experiments.

 

3. Strains designation  and scientific proof of strains identifications must be  presented in section 2.1. Microorganisms. A statement of the availability of these strains from the y H.T.M. Comercio e Laboratórios de Corretivos do Solo Ltda - Biosag 110 (Ituverava, SP, Brazil) must be included.  Without these information, the reproducibility of the presented data (by other scientists) are questionable.

Response:

The fungal isolates are stored at the Tropical Collection Culture of the Andre Tosello Foundation (http://fat.org.br/) under the codes CCT7827 (B. bassiana), CCT7829 (T. asperellum), CCT7828 (M. anisopliae) and CCT 7830 (P. chlamydosporia). Part of the ribosomal RNA gene of the isolates was sequenced with SR6R and LR1 primers (White et al., 1990) aiming to confirm the taxonomical classification.

White, T. J., Bruns, T., Lee, S. J. W. T., & Taylor, J. (1990). Amplification and direct sequencing of fungal ribosomal RNA genes for phylogenetics. PCR protocols: a guide to methods and applications, 18(1), 315-322.

 

4. Manuscript need to be carefully  rewritten, to improve precision and clarity. For example, L259-260, “Concerning M. anisopliae, this fungus has been well explored as biopesticide agent for plant defense…” The correct word that must be used is “protection” and not “defense”.

Response:

We have revised the manuscript to improve precision and clarity. The term 'defense' has been adjusted to 'protection' as suggested (L295), and further revisions were made throughout the text.

 

 

Reviewer 2 Report

Comments and Suggestions for Authors

The manuscript presents a compelling study on the production of in-house enzymatic cocktails using endophytic fungi for the saccharification of corn residues. The novelty lies in the use of Metarhizium anisopliae for β-glucosidase production, coupled with the integration of enzymatic hydrolysis with organosolv-pretreated biomasses. The study effectively demonstrates the potential of these homemade enzymatic blends in contributing to a sustainable biorefinery framework, particularly in enhancing reducing sugar yields. Following are the comments and queries for the improvement of the manuscript.

1. While the novelty of using M. anisopliae is highlighted, the manuscript could better contextualize how this enzymatic cocktail compares with commercial preparations or other fungal strains commonly used in similar applications.

2. The methods are adequately described but lack justification for certain key experimental choices, such as specific pH, temperature, and substrate conditions for enzymatic assays. Including these details would enhance the reproducibility of the study.

3. The results are well-presented, but the analysis could be more comprehensive by comparing the findings with relevant literature or commercial standards. Additionally, providing more in-depth discussions on enzyme stability, substrate specificity, and scalability would elevate the manuscript’s impact.

4. While the study contributes valuable insights into enzymatic hydrolysis and pretreatment, it would benefit from addressing scalability and potential economic implications more thoroughly. For instance, discussions on the cost-effectiveness of the SSF process and organosolv pretreatment are limited.

5. The figures and tables are informative but need clearer annotations and explicit inclusion of error bars or variability measures. This would enhance data transparency and strengthen the conclusions drawn.

6. Are there any considerations regarding the environmental and economic costs of the organosolv pretreatment, given its reliance on ethanol?

Author Response

Reviewer 2:

The manuscript presents a compelling study on the production of in-house enzymatic cocktails using endophytic fungi for the saccharification of corn residues. The novelty lies in the use of Metarhizium anisopliae for β-glucosidase production, coupled with the integration of enzymatic hydrolysis with organosolv-pretreated biomasses. The study effectively demonstrates the potential of these homemade enzymatic blends in contributing to a sustainable biorefinery framework, particularly in enhancing reducing sugar yields. Following are the comments and queries for the improvement of the manuscript.

1.  While the novelty of using M. anisopliae is highlighted, the manuscript could better contextualize how this enzymatic cocktail compares with commercial preparations or other fungal strains commonly used in similar applications.

Response:

The authors appreciate the reviewer suggestion. However, these appointments have already been considered in the section 3.2. (Enzymatic hydrolysis), specifically in the lines L447-470 for other fungal strains “Due to the absence of preceding investigations on enzymatic bioconversions of lignocellulosic residues carried out with enzymatic extracts produced by M. anisopliae, the obtained data were compared to reports of hydrolysis executed with in-house enzymes from other fungal species. For example, Rodrigues et al. [19] employed a crude Aspergillus niger enzyme cocktail in saccharifications of hydrothermal (HP) pretreated sugarcane bagasse and obtained 10.8 g/L of glucose and 16.2% of bioconversion rate. These same authors also pretreated SB by HP-Soda, followed by hydrolysis using an extract produced by an A. niger, G. lucidum and Pleurotus ostreatus consortium and achieved 11.92 g/L of glucose. Pereira et al. [14] analyzed the hydrolytic effectiveness of SB pretreated with ozone using a Myceliophthora thermophila JCP 1–4 extract in an enzymatic load of 10 FPU/g substrate and found 4.19 g/L of glucose. Pereira Scarpa et al. [18] tested a Pycnoporus sanguineus MCA16 enzyme cocktail in the hydrolysis of alkaline pretreated SB and detected 7.32 g/L of glucose, using 260 U of endoglucanase/g cellulose. Lei et al. [54] investigated the potential of an on-site Aspergillus costaricensis LS18 enzyme extract  produced by SSF using lycium barbarum leaves (LBL) as substrate. The saccharification of LBL carried out with this cocktail reached a TRS concentration of 8.17 g/L. Teixeira et al. [55] investigated the role of a T. asperellum PEC-6/P cocktail containing xylanase and β-glucosidase activities in the saccharification of pineapple crown waste and obtained a conversion rate of 13.93%. In the present study, higher and promising yields of cellulose bioconversion were achieved in the hydrolysates of pretreated CS and CC after the hydrolysis with this novel on-site M. anisopliae enzyme solution (Table 5). In addition, superior TRS concentrations were reached in the hydrolysates of organosolv pretreated CS and CC using this innovative M. anisopliae enzymatic extract, in comparison to these earlier studies” and lines L.471-478 for commercial solutions: “The TRS release in pretreated CS and CC hydrolyzed by this M. anisopliae HTM extract were also superior than in some studies carried out with commercial enzymes. For example, Klein et al. [56] evaluated hydrolytic efficiency in H₂SO₄ pretreated banana peel waste employing a commercial cellulase (Sigma-Aldrich) and the highest yield of TRS was of 11.88 g/L and Zhu et al. [57] acquired 13.18 g/L of reducing sugars after the enzymatic saccharification of wheat straw pretreated with ultrasound and dilute alkali cooking (RU), using 20FPU/g residue of cellulase (C2730). These data reinforce the promising role of this M. anisopliae enzyme cocktail in lignocellulose bioconversions”.

Since the focus of this study was to present endophytic fungal strains as novel sources of hydrolytic enzymes, exploring their feasibility and stability, we believe that this work makes a significant contribution to this research field, with emphasis for M. anisopliae β-glucosidase, which can be explored in various biotechnological contexts, particularly in hydrolysis of lignocellulosic substrates. Therefore, this study would be seen as a contribution to the understand the diversity of enzymes produced by these fungal species, providing important insights of prospection and paving the way for future commercial-scale applications.

 

2. The methods are adequately described but lack justification for certain key experimental choices, such as specific pH, temperature, and substrate conditions for enzymatic assays. Including these details would enhance the reproducibility of the study.

Response: We appreciate the reviewer’s recommendation and provided more detailed explanation of the conditions for enzymatic assays (L162-167): “The temperature of 50°C and pH 4.8 were selected for the enzymatic assays based on the commonly conditions applied for hydrolytic processes employing cellulases and hemicellulases, providing a balance between structural stability and catalytic activity [29,30]. Besides, the chosen temperature and pH values fall within the optimal activity range for the evaluated hydrolytic enzymes and reduces the probability of microbiological contamination, improving experimental control [31,32]”.

 

3. The results are well-presented, but the analysis could be more comprehensive by comparing the findings with relevant literature or commercial standards. Additionally, providing more in-depth discussions on enzyme stability, substrate specificity, and scalability would elevate the manuscript’s impact.

Response:

The authors appreciate the reviewer’s suggestions. However, some of these appointments have already been included in the manuscript; for example, the enzyme stability and applicability were addressed in the subsections 2.4 (β-Glucosidase characterization) and 2.6 (Enzymatic hydrolysis). Other issues, such as substrate specificity and scalability, was not the scope of this study, which focused mainly on the originality and specific findings of these novel fungal enzyme preparations. We believe that the presented results provide a comprehensive view of these new on-site cocktails, and at this stage, consider the current data sufficient to support the manuscript’s conclusions.

 

4. While the study contributes valuable insights into enzymatic hydrolysis and pretreatment, it would benefit from addressing scalability and potential economic implications more thoroughly. For instance, discussions on the cost-effectiveness of the SSF process and organosolv pretreatment are limited.

Response:

We agree that scalability and economic implications are important factors for the practical application of enzymatic hydrolysis and substrate pretreatment. However, this study was primarily focused on exploring the technical aspects of enzymatic efficiency and hydrolysis of these novel enzymatic cocktails. Therefore, a detailed analysis of the cost-effectiveness of the SSF process and pretreatment was not included in this work. We recognize the relevance of these aspects for the further development and commercialization of these enzymes and pretend to address them in future studies.

 

5. The figures and tables are informative but need clearer annotations and explicit inclusion of error bars or variability measures. This would enhance data transparency and strengthen the conclusions drawn.

Response:

We have carefully addressed all the issues as requested:

Figures 1,2 and 3 error bars: We have updated the captions for Figures 1 and 2 to include detailed information about the error bars, specifying that they represent the standard deviation of the mean values. Additionally, all data points in figures now include error bars.

Figure 3 caption: As specified in the caption of Figure 3, error bars representing the standard deviation (n = 3) have been added to each data point in the graph.

 

6. Are there any considerations regarding the environmental and economic costs of the organosolv pretreatment, given its reliance on ethanol?

Response:

We appreciate the reviewer’s observation regarding the environmental and economic costs of the employed organosolv pretreatment. Ethanol was chosen since it is a renewable solvent, less harmful to environment and more economical than other chemicals commonly used in pretreatments, such as acids and alkaline reagents. We recognize that a more comprehensive life cycle assessment and cost analysis would be valuable for assessing the sustainability of this approach. Nonetheless, in this first moment, the primary goal was on the efficacy of the new M. anisopliae cocktail in the enzymatic hydrolysis. Future studies will investigate the environmental and economic impacts, considering this one and other kinds of pretreatment and biomasses.

Round 2

Reviewer 1 Report

Comments and Suggestions for Authors

The authors made  improvements. However, there are still important issued that still need be corrected. The authors mentioned in their answer: “The fungal isolates are stored at the Tropical Collection Culture of the Andre Tosello Foundation (http://fat.org.br/) under the codes CCT7827 (B. bassiana), CCT7829 (T. asperellum), CCT7828 (M. anisopliae) and CCT 7830 (P. chlamydosporia). Part of the ribosomal RNA gene of the isolates was sequenced with SR6R and LR1 primers (White et al., 1990) aiming to confirm the taxonomical classification." However, Section 2.1. Microorganisms, L109-113, do not mention these details essential to reproduce their reported data. These info must be included in the article.

They do not mentioned producer of the software (L241-242)

There are still  data without error bars – e.g. CMCase activity vs. time for Pochonia chlamydosporia (Figure 1).

In Figure 1 and Figure 2 the different type of activities (e.g. CMCase, Avicelase, β-glucosidase) need to be differentiated by letter for each graph  and mentioned in figures captions.

The authors did not complete the final statements – Authors Contribution, Conflict  of Interests. Without these Statements the manuscript is not publishable.

Author Response

Please see an attachment.

Author Response File: Author Response.docx