Effect of Aspergillus niger Fermentation on the Metabolites in Corn Stalks
, Xiaoyu Huang 1, Suchuan Zhong 1, Xiaoming Li 2 and Enping Zhang 1,*Round 1
Reviewer 1 Report
The manuscript describes the Aspergillus niger-mediated fermentation of corn stalks and identifies metabolites released in the process. The authors have applied cutting-edge analytics and tools to identify and associate metabolites to the KEGG pathway. Although the general workflow is convincing, using only one sample each of treated and untreated corn stalks could be limiting since this precludes the evaluation of both biological variability associated with the organism and technical issues with sampling or handling. Besides this, the following points need clarification or correction.
1- I do not think GS-MS should be part of the title.
Line 60 -61: The possible effects of the "differential metabolites" "on growth performance and immune responses" have not been evaluated or discussed convincingly in this manuscript.
Line 79: What informed the duration of the fermentation? Could shorter or longer fermentation be more beneficial?
Line 85: Please briefly describe the Singh et al. method and any modifications applied in the current work.
Line 114 -116: Provide a detailed description of the methods employed, including tools and algorithms, parameter settings and selection of background KEGG compounds and the strategy used in associating metabolites to metabolic pathways.
Line 123: NDF in full here.
Line 128: Reference is inadequate here since none of the above activities or enzymes specifically address Aspergillus niger. See https://biotechnologyforbiofuels.biomedcentral.com/articles/10.1186/s13068-017-0700-9 for example. Please check all other references for accuracy.
Table 1. Natural detergent fibre or Neutral detergent fibre
Section 3.2: This may be moved to supplementary as it appears irrelevant as a standalone subsection.
Line 170 -173: Long and difficult to follow. Screen out means 370 'differential metabolites' were removed from the analysis.
Line 173 -175: I am particularly unconvinced about using the terms up- and down-regulated in this manuscript since these have special meaning in expression or 'omics' studies. It's also on the borderline of misleading the reader about the precise experiment done. Consider the term overrepresented (increase) and underrepresented (decrease) instead. Also, the authors should highlight the relevant conditions to give some context to the data, for instance, the significant overrepresentation of 172 metabolites in Fer relative to Con.
Figure 3: Rephrase the title and legend to reflect the above comments on using inappropriate terminology and amend throughout the manuscript.
Line 206 -210: Too long and confusing sentence.
Line 214 -219: Cite source as appropriate.
Line 218: Please verify that this statement is correct.. the two sections of the sentence are not related and are confusing.
Line 221 -229: Please cite sources as appropriate.
1- the positive effect on intestinal flora and 2- the impact of N-acetylgalactosamine. On the latter, the authors need to review relevant literature on the putative toxicity of NAcGal and its threshold relative to values reported in this study.
Lines 227 -229: The statement is too generic and needs context. What is carbohydrate metabolism pathway?
Line 232-233: Statement too generic and not supported by the cited reference.
Line 233 -236: Sentence incomplete.
Line 236 -239: Unsupported statement.
Line 243 -246: Not clear whether the authors are reporting their work or discussing previous findings.
Line 246 -251: Please carefully edit the text to prune irrelevant expressions and cite sources as appropriate.
Line 256: Please refer to the relevant table or figure and also throughout the manuscript as appropriate.
Line 260-264: Statement confusing. "The down-regulation of isochlorogenic acid and chlorogenic acid mainly considers that the temperature during fermentation of Aspergillus may lead to a certain degree of degradation, or it may be various enzymes produced by microbial fermentation, including complex enzymes (cellulase, lacquer, xylanase, etc.) with high catalytic activity".
Line 266 -271: "mostly due to the consumption of biosynthesis in the process of microbial metabolism" Could the authors clarify this? Also, neither clear reasoning nor literature supports the discussion.
Line 277 -279: More informative to focus on the one amino acid that increased with Aspergillus fermentation.
Line 286 -289: Completely non-informative and possibly erroneous statement. “metabolic pathways such as non-oxidative decarboxylation reaction and other metabolic pathways in the cell wall of raw and auxiliary materials during the fermentation process of Aspergillus”: Please clarify.
Line 295: Unsupported statement.
3.3.8: Please improve the accuracy of the information and cite the relevant literature.
Section 3.4: First and foremost, this section is the most ambiguous.
1- It is entirely appropriate to determine metabolites differences between treated and untreated stalks.
2- Associating the observed metabolites with relevant pathways also makes sense.
3- This analysis should be carried out with utmost caution to avoid confusion.
4- The listed pathways are not "involved in significantly differential metabolites", as stated by the authors. See lines 324 - 325
5- ko02060 is Phosphotransferase system (PTS), not "the central carbon metabolism pathway in cancer". If any studies have linked this pathway with cancer, discuss such studies, but don't rename any KEGG pathway.
6- ko00040 is pentose and glucuronic interconversions. Note the 'and'.
7- The statement in lines 236 -234 is unfounded. The presented experimental design precludes any inference about the "improvement of the nutritional value of A. niger cultures" or the implications thereof.
8- Did the authors consider all identified metabolites or only metabolites showing a significant increase or decrease after Aspergillus fermentation?
9- Did the authors use all metabolites associated with the KEGG PATHWAY of Aspergillus niger as a reference?
10- Do 120 compounds provide sufficient background metabolite set for computing or speculating the enrichment of any particular pathway?
Finally, the objectives behind the pathway analysis are unclear. Therefore, motivate and discuss the results with relevant literature.
Author Response
Thank you very much for reviewing our manuscript. We have read the constructive suggestions from you and carefully revised the manuscript accordingly.
- I do not think GS-MS should be part of the title.
Response:
The title has been revised, and GC-MS has been deleted.
Line 60 -61: The possible effects of the "differential metabolites" "on growth performance and immune responses" have not been evaluated or discussed convincingly in this manuscript.
Response:
The sentence has been revised. (Lines 61-62, in red)
Line 79: What informed the duration of the fermentation? Could shorter or longer fermentation be more beneficial?
Response:
We optimized the fermentation time with the degradation rate of NDF and ADF. We found that longer fermentation (6 days) is more beneficial than shorter (3, 4 and 5 days).
Line 85: Please briefly describe the Singh et al. method and any modifications applied in the current work.
Response: Lines 91-104, in red
Neutral detergent fiber (NDF) was determined by the national standard of the People’s Republic of China (GB/T 20806-2006), and acid detergent fiber (ADF) was determined by the agricultural industry standard of the People’s Republic of China (NY/T 1459-2022). For the hemicellulose determination, the sample is extracted with cold-water, hot-water and alcohol step by step, and the residue is treated by 2% dilute hydrochloric acid. The sugar in the extract is determined, and the the hemicellulose content was calculated. In case of cellulose, the residue above is treated with 10 volumes of sulfuric acid, and then autoclaved at 121°C for 1 hour after 15 volumes of water was added. Followed by neutralized with 10% sodium hydroxide solution and filtering, the sugar is determined. The residue from the above sulfuric acid treatment are thoroughly washed, dried, and weighed. The lignin content was calculated by minus the ash and crude protein content according to ordinary methods.
Line 114 -116: Provide a detailed description of the methods employed, including tools and algorithms, parameter settings and selection of background KEGG compounds and the strategy used in associating metabolites to metabolic pathways.
Response: Lines 138-145, in red
Differential metabolites among two groups were summarized and mapped into their biochemical pathways through metabolic enrichment and pathway analysis based on database search (KEGG, http://www.genome.jp/kegg/). These metabolites can be classified according to the pathways they involved or the functions they performed. Enrichment analysis was usually to analyze a group of metabolites in a function node whether appears or not. Enrichment analysis of KEGG pathways was performed using Fisher’s exact test. Statistical analyses were performed using R software v3.6.2.
Line 123: NDF in full here.
Response: It has been revised. Lines 152-153, in red
Line 128: Reference is inadequate here since none of the above activities or enzymes specifically address Aspergillus niger. See https://biotechnologyforbiofuels.biomedcentral.com/articles/10.1186/s13068-017-0700-9 for example. Please check all other references for accuracy.
Response: Thank you for carefully review, it has been revised. Line 157, in red
Table 1. Natural detergent fibre or Neutral detergent fibre
Response:We are very sorry for this mistake. It is Neutral detergent fiber. Table 1, in red
Section 3.2: This may be moved to supplementary as it appears irrelevant as a standalone subsection.
Response: Thank you for your advised, and the section 3.2 has been moved to supplementary.
Line 170 -173: Long and difficult to follow. Screen out means 370 'differential metabolites' were removed from the analysis.
Response: The sentence has been revised, and “screen out” was changed with “identified”. Line 187, in red
Line 173 -175: I am particularly unconvinced about using the terms up- and down-regulated in this manuscript since these have special meaning in expression or 'omics' studies. It's also on the borderline of misleading the reader about the precise experiment done. Consider the term overrepresented (increase) and underrepresented (decrease) instead. Also, the authors should highlight the relevant conditions to give some context to the data, for instance, the significant overrepresentation of 172 metabolites in Fer relative to Con.
Response: Thank you for your constructive advice, the terms increase and decrease were used. The fold change was used as relevant conditions to distinguish the increase or decrease metabolites. Lines 188-190, in red
Figure 3: Rephrase the title and legend to reflect the above comments on using inappropriate terminology and amend throughout the manuscript.
Response: It has been revised.
Line 206 -210: Too long and confusing sentence.
Response: It has been revised. Lines 219-222, in red
Line 214 -219: Cite source as appropriate.
Response: It has been revised. Lines 227-228, in red
Line 218: Please verify that this statement is correct.. the two sections of the sentence are not related and are confusing.
Response: It has been revised. Lines 229-230, in red
Line 221 -229: Please cite sources as appropriate.
1- the positive effect on intestinal flora and
2- the impact of N-acetylgalactosamine. On the latter, the authors need to review relevant literature on the putative toxicity of NAcGal and its threshold relative to values reported in this study.
Response: The appropriate reference has been cited, and the irrelevant expressions were deleted as there isn’t relevant literature on the toxicity threshold of NAcGal. Lines 232-236, in red
Lines 227 -229: The statement is too generic and needs context. What is carbohydrate metabolism pathway?
Response: The sentence has been revised, and “and various metabolites were formed through the carbohydrate metabolism pathway” was deleted. Lines 236-237, in red
Line 232-233: Statement too generic and not supported by the cited reference.
Response: It has been revised. Lines 240-241, in red
Line 233 -236: Sentence incomplete.
Response:The sentence has been revised. Lines 241-244, in red
Line 236 -239: Unsupported statement.
Response: The appropriate reference has been cited. Line 245, in red
Line 243 -246: Not clear whether the authors are reporting their work or discussing previous findings.
Response: The sentence has been revised. Lines 251-253, in red
Line 246 -251: Please carefully edit the text to prune irrelevant expressions and cite sources as appropriate.
Response: The irrelevant expressions were deleted and the appropriate reference has been cited. Lines 255, in red
Line 256: Please refer to the relevant table or figure and also throughout the manuscript as appropriate.
Response: It has been revised.
Line 260-264: Statement confusing. "The down-regulation of isochlorogenic acid and chlorogenic acid mainly considers that the temperature during fermentation of Aspergillus may lead to a certain degree of degradation, or it may be various enzymes produced by microbial fermentation, including complex enzymes (cellulase, lacquer, xylanase, etc.) with high catalytic activity".
Response: The sentence has been revised and the appropriate reference has been cited. Lines 265-266, in red
Line 266 -271: "mostly due to the consumption of biosynthesis in the process of microbial metabolism" Could the authors clarify this? Also, neither clear reasoning nor literature supports the discussion.
Response: It is just our hypothesis based on the results and the sentence has been revised. Lines 270-274, in red
Line 277 -279: More informative to focus on the one amino acid that increased with Aspergillus fermentation.
Response: The irrelevant expressions were deleted.
Line 286 -289: Completely non-informative and possibly erroneous statement. “metabolic pathways such as non-oxidative decarboxylation reaction and other metabolic pathways in the cell wall of raw and auxiliary materials during the fermentation process of Aspergillus”: Please clarify.
Response: The sentence has been revised. Vanillin is a type of aroma substance formed by bio-transformation, such as the non-oxidative decarboxylation reaction of ferulic acid to 4-vinyl guaiacol and then converted into vanillin during the fermentation process of Aspergillus. Lines 287-290, in red
Line 295: Unsupported statement.
Response: The appropriate reference has been cited. Lines 297, in red
3.3.8: Please improve the accuracy of the information and cite the relevant literature.
Response: The accuracy of the information has been improved, and the appropriate reference has been cited. Line 308, 311, 314, in red
Section 3.4: First and foremost, this section is the most ambiguous.
1- It is entirely appropriate to determine metabolites differences between treated and untreated stalks.
2- Associating the observed metabolites with relevant pathways also makes sense.
3- This analysis should be carried out with utmost caution to avoid confusion.
4- The listed pathways are not "involved in significantly differential metabolites", as stated by the authors. See lines 324 - 325
5- ko02060 is Phosphotransferase system (PTS), not "the central carbon metabolism pathway in cancer". If any studies have linked this pathway with cancer, discuss such studies, but don't rename any KEGG pathway.
6- ko00040 is pentose and glucuronic interconversions. Note the 'and'.
7- The statement in lines 236 -234 is unfounded. The presented experimental design precludes any inference about the "improvement of the nutritional value of A. niger cultures" or the implications thereof.
8- Did the authors consider all identified metabolites or only metabolites showing a significant increase or decrease after Aspergillus fermentation?
9- Did the authors use all metabolites associated with the KEGG PATHWAY of Aspergillus niger as a reference?
10- Do 120 compounds provide sufficient background metabolite set for computing or speculating the enrichment of any particular pathway?
Finally, the objectives behind the pathway analysis are unclear. Therefore, motivate and discuss the results with relevant literature.
Response: The section was revised, and the observed metabolites were associated with relevant pathways. Lines 319-337, in red
Author Response File: 
Author Response.pdf
Reviewer 2 Report
Your paper dealing with identifcation of different compounds produced by working microorganism and influence of its metabolism on changes in compositon of raw material in order to improve the nutritional quality of animal feed has interesting approach.
But unfortunately, there are certain issues that need detailed clarification and modification in experimental/analytical approach.
Materials and Methods section needs rewriting (see comments). Results and Discussion section contains data that should be placed in M&M or supplementary material. And R&D section should be rewritten completely to give better understanding of obtained results.
Additionally, English language in this paper needs improvement. Reference style should match journal instructions. Figures have poor resolution. From Fig 4. it is impossible to see the compounds determined by analysis.
There are two Table 1 in this paper (Table 1. Effect of A. niger fermentation on the composition of corn stalks (% DM). vs Table 1. 32 differential characteristic metabolites associated with Aspergillus fermentation.)
Comments:
L35 "Silage as an anaerobic digestion technology..." Silage is the product, not the technology. Technology for production of silage is called ensiling.
L37 same comment as in L35
L39 Aspergillus is a genus, not a strain...
L53-L55 Please rewrite, unclear
L64 Material and methods section needs detailed rewriting
For all chemicals used in research a manufacturer and state should be given (e.g. Merck, Darmstadt, Germany).
Methodology of inoculum and media preparation is not described sufficiently. Some questions:
1)This A. niger HQ2 strain was selected, please show the results or give reference...
2) water content of used corn stalks should be given
3) how was supplementation of salts performed? where they dissolved in
water? what kind of water was used?
how did u prepare spore suspension? in what did u suspend spores?
4) The sentence "after fermentation..." is unclear. It is method of sample preparation, explain more thoroughly
L82-L85 Biochemical analysis
Here u refer to Singh (reference 14) where when u open Singh paper it leads u to another paper by Waksman and Stevens from 1930 where several methods for cellulose, hemicellulose, lignin, protein and ash content are described. Therefore, the methods used in this work should be shortly describe in this paragraph and cited.
L95 Please add derivatization procedure used, this is a very important step in your experiment due to GC-MS identification of compounds.
Derivatiozation procedure here can be drawback to your identification of fermentation compounds.
L95 Name the GC-MS model and producer of this equipment
L120 Results and discussion section should contain, besides results, also a discussion. There is no comparison of your data with data available in literature, no reference has been used from L120-L198.
L127 u are assuming the activity of cellulolytic enzymes, that can be analytically confirmed
L129 based on what do u conclude this?
Table1 Abbreviation not explained %DM, dry matter %
L134-L136 This is not a result, it is a standard procedure used in chromatographic analysis
L137 what are the QC samples, no explanation before in text
Figure 1 should not be a part of the paper, it suits better in supplementary material
L152 what are the metabolites detected, there is no mention before in text, or it can be seen from the fig 2A
Author Response
Response to Reviewer 2 Comments
Thank you very much for reviewing our manuscript. We have read the constructive suggestions from you and carefully revised the manuscript accordingly. The revised manuscript has been proofed with the assistance from an MDPI English editing.
Your paper dealing with identifcation of different compounds produced by working microorganism and influence of its metabolism on changes in compositon of raw material in order to improve the nutritional quality of animal feed has interesting approach.
But unfortunately, there are certain issues that need detailed clarification and modification in experimental/analytical approach.
Materials and Methods section needs rewriting (see comments). Results and Discussion section contains data that should be placed in M&M or supplementary material. And R&D section should be rewritten completely to give better understanding of obtained results.
Additionally, English language in this paper needs improvement. Reference style should match journal instructions. Figures have poor resolution. From Fig 4. it is impossible to see the compounds determined by analysis.
There are two Table 1 in this paper (Table 1. Effect of A. niger fermentation on the composition of corn stalks (% DM). vs Table 1. 32 differential characteristic metabolites associated with Aspergillus fermentation.)
Comments:
L35 "Silage as an anaerobic digestion technology..." Silage is the product, not the technology. Technology for production of silage is called ensiling.
Response: Thank you, it has been revised accordingly. Line 35, in red
L37 same comment as in L35
Response: It has been revised accordingly. Line 37, in red
L39 Aspergillus is a genus, not a strain...
Response: Thank you, it has been revised accordingly. Line 39, in red
L53-L55 Please rewrite, unclear
Response: The sentence has been rewrote. Lines 54-57, in red
L64 Material and methods section needs detailed rewriting
Response: It has been revised accordingly. Lines 67-74, 78-89, 91-104, 112-116, 118-119, in red
For all chemicals used in research a manufacturer and state should be given (e.g. Merck, Darmstadt, Germany).
Response: Thank you, all chemicals used in research has been revised accordingly. Lines 67-74, in red
Methodology of inoculum and media preparation is not described sufficiently. Some questions: Lines 78-89, in red
1) This A. niger HQ2 strain was selected, please show the results or give reference...
Response: The A. niger HQ2 strain was isolated in corn stalks and preserved in our lab, and the data didn’t show yet.
2) water content of used corn stalks should be given
Response: The dried corn stalk was used in this study, and its moisture is 10.2%.
3) how was supplementation of salts performed? where they dissolved in water? what kind of water was used? how did u prepare spore suspension? in what did u suspend spores?
Response: Except for sodium chloride and calcium chloride dissolved in deionized water, other substances are directly mixed with corn stalks evenly. The strain A. niger HQ2 was activated in PDA (30 °C, 5 d), and Tween 80 solution (0.01%) was used to recover the spores. The spores were calculated by counting with Neubauer’s chamber, and were adjusted to 108 spores/mL for further use.
4) The sentence "after fermentation..." is unclear. It is method of sample preparation, explain more thoroughly
Response: After fermentation, the sample was placed in a 60°C oven to dry to a moisture content of about 12%, and pulverize to a 40-mesh pass rate of over 80% for use.
L82-L85 Biochemical analysis
Here u refer to Singh (reference 14) where when u open Singh paper it leads u to another paper by Waksman and Stevens from 1930 where several methods for cellulose, hemicellulose, lignin, protein and ash content are described. Therefore, the methods used in this work should be shortly describe in this paragraph and cited.
Response: Lines 91-104, in red
The methods used in this work has been described and cited appropriately. Neutral detergent fiber (NDF) was determined by the national standard of the People’s Republic of China (GB/T 20806-2006), and acid detergent fiber (ADF) was determined by the agricultural industry standard of the People’s Republic of China (NY/T 1459-2022). For the hemicellulose determination, the sample is extracted with cold-water, hot-water and alcohol step by step, and the residue is treated by 2% dilute hydrochloric acid. The sugar in the extract is determined, and the the hemicellulose content was calculated. In case of cellulose, the residue above is treated with 10 volumes of sulfuric acid, and then autoclaved at 121°C for 1 hour after 15 volumes of water was added. Followed by neutralized with 10% sodium hydroxide solution and filtering, the sugar is determined. The residue from the above sulfuric acid treatment are thoroughly washed, dried, and weighed. The lignin content was calculated by minus the ash and crude protein content according to ordinary methods.
L95 Please add derivatization procedure used, this is a very important step in your experiment due to GC-MS identification of compounds. Derivatiozation procedure here can be drawback to your identification of fermentation compounds.
Response: Lines 112-116, in red
The derivatization procedure has been added. Methoxyamine hydrochloride pyridine solution (15 mg/mL, 80 μL) was added and then oximation reaction was performed at 37 °C for 90 min. A volume of 50 μL BSTFA (containing 1% TMCS) derivatization reagent and 20 μL N-hexane were added into the samples, and then the internal standards were added subsequently to react at 70 °C for 60 min.
L95 Name the GC-MS model and producer of this equipment
Response: The derivatived samples were analyzed on an Agilent 7890B gas chromatography system coupled to an Agilent 5977B MSD system (Agilent Technologies Inc., CA, USA). Lines 118-119, in red
L120 Results and discussion section should contain, besides results, also a discussion. There is no comparison of your data with data available in literature, no reference has been used from L120-L198.
Response: the section 3.2 has been moved to supplementary.
L127 u are assuming the activity of cellulolytic enzymes, that can be analytically confirmed
Response: Thank you, we will detect the activity of all carbohydrate-active enzymes in the next.
L129 based on what do u conclude this?
Response: The sentence has been revised. Line 160, in red
Table1 Abbreviation not explained %DM, dry matter %
Response: It has been revised. Line 162, in red
L134-L136 This is not a result, it is a standard procedure used in chromatographic analysis
Response: It has been moved to supplementary material.
L137 what are the QC samples, no explanation before in text
Response: the section 3.2 has been moved to supplementary.
Figure 1 should not be a part of the paper, it suits better in supplementary material
Response: It has been moved to supplementary material.
L152 what are the metabolites detected, there is no mention before in text, or it can be seen from the fig 2A
Response: It has been revised. Line 168, in red
Author Response File: Author Response.pdf
Round 2
Reviewer 1 Report
The current version of the manuscript represents an improvement over the earlier version.
However, the description of enrichment analysis in lines 139 -40 is incorrect. The results of the metabolic pathway analysis remain undiscussed. Although figure 5 includes relevant information, the text in 316 - 334 is simply a list of top 20 "overrepresented pathways" without any context.
Author Response
The current version of the manuscript represents an improvement over the earlier version. However, the description of enrichment analysis in lines 139 -40 is incorrect
Response: The sentence has been revised.
Pathways with significantly changed metabolites mapped to were then fed into metabolite sets enrichment analysis. Enrichment analysis of KEGG pathways was performed using Fisher’s exact test. (Lines 144-145, in red)
The results of the metabolic pathway analysis remain undiscussed. Although figure 5 includes relevant information, the text in 316 - 334 is simply a list of top 20 "overrepresented pathways" without any context.
Response: Thanks for your constructive advice, and we have added some information about metabolic pathways (Lines 331-332, 335-337).
Reviewer 2 Report
After reading manuscript,
there are only minor issues to correct:
L112 please name the internal standards used
Throughout the text please check the writing of chemical compounds like in "N-acetyl-d-mannosamine" (e.g. small/capital letter, italic...)
Author Response
After reading manuscript, there are only minor issues to correct:
L112 please name the internal standards used.
Response: Ten fatty acid methyl esters and L-2-chlorophenylalanine were used as the internal standards (Lines 116-117, in red). The information of the internal standards is described in Lines 73-76.
Throughout the text please check the writing of chemical compounds like in "N-acetyl-d-mannosamine" (e.g. small/capital letter, italic...)
Response：Thanks for carefully reviewed, and we revised them accordingly. (Line 219, 220, 240)
