Agro-Industrial Wastes: A Substrate for Multi-Enzymes Production by Cryphonectria parasitica

Round 1

Reviewer 1 Report

The paper aimed the evaluation of feasibility of different food wastes as sole substrates, with no supplements, concerning the the growth and the production of aspartic endopeptidase (EC 3.4.23), carboxylesterase and laccase (EC 1.10.3.2) by environmental C. parasitica strains under SSF. 

The methodology is complex and complete, the results and discussion is also consistent, also supported by citing references. Still, the Conclusion section needs improvement. Please address Conclusion through your obtained results, not by general aspects. 

Author Response

Answer Point by point answer to reviewer

Comment: The paper aimed the evaluation of feasibility of different food wastes as sole substrates, with no supplements, concerning the the growth and the production of aspartic endopeptidase (EC 3.4.23), carboxylesterase and laccase (EC 1.10.3.2) by environmental C. parasitica strains under SSF. 

The methodology is complex and complete, the results and discussion is also consistent, also supported by citing references. Still, the Conclusion section needs improvement. Please address Conclusion through your obtained results, not by general aspects. 

Answer: As suggested by the reviewer the conclusion section was revised and now reads as

The ability of the filamentous ascomycete C. parasitica to colonize agro-industrial wastes without any pre-treatment and supplements as a source of value-added bio-products by means of SSF was verified in this study.

To the best of our knowledge, this is the first report on multiple enzymes production by C. parasitica on agro-industrial wastes. All newly isolated C. parasitica strains were able to i) grow, metabolize the substrate, and ii) produce multiple enzymes on ZW without any other supplement. Interestingly, the crude extract obtained from the hypovirulent strain CpC4 showed the highest enzymatic activities, resulting an ideal candidate for further studies on the feasibility of large-scale production of industrially relevant enzymes.

In the circular economy view, the here presented approach could lead to develop a biorefinery without waste production. The fermentation substrates could be addressed for the production of microorganism-based fertilizer.

Reviewer 2 Report

Chestnut blind is a fastidious disease that affects crops throughout Europe and is responsible for the economic loss of many industries dedicated to this cultivar. For this reason, I consider that this work may be of interest, however the writing of the manuscript is not adequate, the lack of cohesion of the sentences makes it unattractive. I recommend that the authors review and read the manuscript carefully, they need to reorganize the information, reduce the incongruity of the statements, apply statistical treatments to see the significant differences and avoid talking about data not shown. In the current form it is difficult to assess whether the information is valid or not.

General comments:

The manuscript needs a thorough revision of English, preferably by a native speaker, due to there are paragraphs that need to be rewritten. Throughout the text to many repeated words and infrequent terms are used. There is also no consistency when using decimal separators. Already in the abstract you can see a decimal with a point and another with a comma.

Specific comments:

• Abstract: “Cryphonectria parasitica (Murr.) Barr. environmental strains” This is the correct name of the strain? Environmental strains?
• Introduction line 64 “SF has a lower energy consumption” I do not understand in what sense, could you clarify this statement?
• Introduction line 75 Please be consistent with the strain name “Cryphonectria parasitica (Murrill) M.E. Barr”
• Why Chestnut Blight is capitalized?
• Introduction line 75-82 Please verify this information within the FDA page, I cannot find information about it and I find it strange that a pathogen is considered GRASS by this organization.
• Please improve the final paragraph of the introduction.

• Materials and methods, lines 92-93: the Agri-food  and  Environmental Microbiology  Platform is an official collection? I can not find nothing about it.
• Materials and methods line 96, Why do the authors use light to grow fungi?
• Materials and methods lines 100-106, I have not read anything about the relevance of this virus neither in the abstract nor in the intro, why does it seem important now? Maybe the authors should be explained something before.
• Materials and methods: The data of Table 1 are results or not? Should they move to another section?
• Materials and methods section 2.2.1: What is zootechnical grade wheat bran? Perhaps the authors mean that it is animal feed?
• Materials and methods, section 2.2.1 “Local stores and bars” The raw materials used were collected from bars at the same time of production or were they stored for weeks? The storage of lignocellulosic material in uncontrolled environments can lead to the proliferation of other organisms and to alter the structure and composition of the material.
• Table 2, again results in the middle of materials and methods... “Approximate chemical composition” Approximate? Where is the standard desviation? It is mandatory to repeat the experiments at least in duplicate. The composition percentages exceed 100% and it is impossible. This table also does not show moisture contents.
• Results and discussion line 218 “It is well-known that the different residues used as substrate can modify fungi gene expression” Any reference?
• Results and discussion Line 222: At this point I still do not know what the moisture contents studied were. This manuscript does not show an optimization of a process, but rather shows the results of 3 moisture conditions.
• Figures 1 and 2 are not clearly visible and do not add anything to the manuscript. The growth of the fungus should be studied quantitatively (weight for example) and not visually.
• Most of the results are accompanied by “data not shown”, so they are not valid and so the manuscript loses interest.
• Line 296 “pesticide residues in the ZW” Pesticides in waste provided by local stores and bars? Really?
• At this point I am completely lost in the manuscript, it has lost all coherence.

Author Response

Reviewer 2

Comment: Chestnut blind is a fastidious disease that affects crops throughout Europe and is responsible for the economic loss of many industries dedicated to this cultivar. For this reason, I consider that this work may be of interest, however the writing of the manuscript is not adequate, the lack of cohesion of the sentences makes it unattractive. I recommend that the authors review and read the manuscript carefully, they need to reorganize the information, reduce the incongruity of the statements, apply statistical treatments to see the significant differences and avoid talking about data not shown. In the current form it is difficult to assess whether the information is valid or not.

Answer: as suggested by the reviewer we extensively revised the manuscript avoiding repetitions and reducing the data not shown.

General comments:

The manuscript needs a thorough revision of English, preferably by a native speaker, due to there are paragraphs that need to be rewritten. Throughout the text to many repeated words and infrequent terms are used. There is also no consistency when using decimal separators. Already in the abstract you can see a decimal with a point and another with a comma.

Answer As you can see in the new version, the manuscript has been extensively re-written, we extensively revised also the language.

Specific comments:

• Abstract: “Cryphonectria parasitica(Murr.) Barr. environmental strains” This is the correct name of the strain? Environmental strains?

Answer: as suggested by the reviewer the name of the Cryphonectria parasitica isolates was specified in the abstract

• Introduction line 64 “SF has a lower energy consumption” I do not understand in what sense, could you clarify this statement?

Answer: Solid state fermentation required lower energy related to fermentation process inputs (oxygen, nutrients) and than submerged fermentation. For clarity the sentence was rephrased adding a new reference.

• Introduction line 75 Please be consistent with the strain name “Cryphonectria parasitica(Murrill) M.E. Barr”

Answer: as suggested by the reviewer we standardize the strain name as Cryphonectria parasitica (Murr.) Barr

• Why Chestnut Blight is capitalized?

Answer: the capital letters were eliminated

• Introduction line 75-82 Please verify this information within the FDA page, I cannot find information about it and I find it strange that a pathogen is considered GRASS by this organization.

Answer: as suggested by the reviewer we verified the information. On the FDA page is reported that some enzymes produced by C. parasitica are considered as GRAS food additives. We revised the manuscript according to it and added the reference.

Answer: as suggested by the reviewer we verified the information. On the FDA page is reported that some enzymes produced by C. parasitica are considered as GRAS food additives. We revised the manuscript according to it and added the reference. Now it reads as ‘Cryphonectria parasitica (Murr.) Barr is a filamentous ascomycete well known for its ability to secrete a range of proteins with industrial applications that are being commercially produced through different fermentation procedures [29–32]. For example milk-clotting enzymes were produced by C. parasitica and they are considered as GRAS (generally recognized as safe) substances [33]’

• Please improve the final paragraph of the introduction.

Answer: The final paragraph of the introduction was enriched with more information about the mycovirus effect on enzyme production 

• Materials and methods, lines 92-93: the Agri-food  and  Environmental Microbiology  Platform is an official collection? I can not find nothing about it.

Answer:  This is a university collection not a commercial one. No need to have a website or similar

• Materials and methods line 96, Why do the authors use light to grow fungi?

Answer: C. parasitica was grown in light condition to induce sporulation and to have a growth condition similar to the the natural ones. vedi https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7565922/

• Materials and methods lines 100-106, I have not read anything about the relevance of this virus neither in the abstract nor in the intro, why does it seem important now? Maybe the authors should be explained something before.

Answer: as reported above we add more information about the relevance of the mycovirus both in the introduction and in the discussion

• Materials and methods: The data of Table 1 are results or not? Should they move to another section?

Answer: Table 1 contains both material both results. As reported in the authors’ instruction, we insert table close to the its first citation in the text.

• Materials and methods section 2.2.1: What is zootechnical grade wheat bran? Perhaps the authors mean that it is animal feed?

Answer: As suggested by the reviewer, zootechnical wheat bran is the animal feed. The sentence was rephrased and now reads as ‘Wheat bran for human (organic wheat bran, OW), or for animal consumption (zootechnical wheat bran, ZW), rice husk (RH), bought at local stores, and spent espresso coffee grounds (CG) collected from bars after espresso preparation in Brescia, Italy, were the by-products used as substrates for fungal growth and enzymes production’

• Materials and methods, section 2.2.1 “Local stores and bars” The raw materials used were collected from bars at the same time of production or were they stored for weeks? The storage of lignocellulosic material in uncontrolled environments can lead to the proliferation of other organisms and to alter the structure and composition of the material.

Answer: we agree with the reviewer: the storage is an important parameter that should be taken into account. The spent coffee ground was collected at the same time of production immediately used as SSF substrate.  A sentence was added in the text: CG were collected after coffee production and immediately used for SSF

• Table 2, again results in the middle of materials and methods... “Approximate chemical composition” Approximate? Where is the standard desviation? It is mandatory to repeat the experiments at least in duplicate. The composition percentages exceed 100% and it is impossible. This table also does not show moisture contents.

Answer: Table 2 doesn’t contain experimental results. The chemical composition of the substrates was deduced from scientific literature. We added these information to better discuss the results. For more clarity the table was moved in the results and discussion section.

• Results and discussion line 218 “It is well-known that the different residues used as substrate can modify fungi gene expression” Any reference?

Answer: we add reference these references

Wu, B.; Gaskell, J.; Held, B.W.; Toapanta, C.; Vuong, T. V; Ahrendt, S.; Lipzen, A.; Zhang, J.; Schilling, J.S.; Master, E.; et al. Retracted and Republished from: “Substrate-Specific Differential Gene Expression and RNA Editing in the Brown Rot Fungus Fomitopsis pinicola” Appl. Environ. Microbiol. 2021, 87, e00329-21, doi:10.1128/AEM.00329-21.

Shang, J.; Wu, X.; Lan, X.; Fan, Y.; Dong, H.; Deng, Y.; Nuss, D.L.; Chen, B. Large-scale expressed sequence tag analysis for the chestnut blight fungus Cryphonectria parasitica. Fungal Genet. Biol. 2008, 45, 319–327, doi:https://doi.org/10.1016/j.fgb.2007.11.002.

• Results and discussion Line 222: At this point I still do not know what the moisture contents studied were. This manuscript does not show an optimization of a process, but rather shows the results of 3 moisture conditions.

Answer: As noticed by the reviewer this is not an optimization of the fermentation parameters that would be performed in further studies including the moisture content, by Response surface methodology. This was just a set up of a protocol allowing contemporarily the production of fungal biomass and/or of a cocktail of industrially relevant enzymes. We compared the fungal growth and the enzyme production obtained from substrates that required the addition of different water content.

• Figures 1 and 2 are not clearly visible and do not add anything to the manuscript. The growth of the fungus should be studied quantitatively (weight for example) and not visually.

Answer: we agree with the reviewer: the fungal biomass should be quantified and we are setting up a protocol for quantifying it in SSF by PCR. As the main goal of this manuscript is the multiple enzyme production we reduced the focus on fungal growth. We replace the figure 1 with a new one with higher resolution and we moved figure 2 in the supplementary material

• Most of the results are accompanied by “data not shown”, so they are not valid and so the manuscript loses interest.

Answer: the results and discussion section was extensively revised and we added supplementary materials to support our data

• Line 296 “pesticide residues in the ZW” Pesticides in waste provided by local stores and bars? Really?

Answer: as the reviewer knows pesticides residues could be present in food and feed at different concentrations, depending on the respective different thresholds. The legal concentrations could still disturb the growth of very sensible isolates. To be certain  to avoid this problem we tested an organic wheat bran (supposed to be pesticides residues free) and a commercial one used for animal feed (supposed to have an higher content of resides). For better clarity we rephrase the sentence that now reads as The potential presence of pesticide residues in the ZW, below the commercial threshold, but still able to interfere with the fungal growth, did not occurred. Thus, even if the organic wheat bran OW gave a faster colonization, ZW was the economical choice as the sole substrate for further experiments of multiple enzymes production through SSF.

• At this point I am completely lost in the manuscript, it has lost all coherence.

Answer: the manuscript was extensively revised both in the English language and in the results presentation and discussion

Round 2

Reviewer 2 Report

The readability and the quality of the manuscript has greatly improved. Besides, the authors have responded satisfactorily to my questions, so I consider the manuscript could be published in the present format.

Author Response

Dear reviewer,

we would like to thank you for the time dedicated to our manuscript. Thanks to his/her critical revision, the present format better valorizes our results.

Finally we revised the manuscript and some minor misspelling were corrected