Utilization of Corncob Biochar in Cultivation Media for Cordycepin Production and Biomass of Cordyceps militaris
, Waraporn Aiphuk 3, Tharakorn Maneerat 4,5,6, Thitipone Suwunwong 4,5, Patcharanan Choto 4,5 and Putarak Chomnunti 3,4,*
Round 1
Reviewer 1 Report
This study investigated effects of medium components on cordycepin and adenosine production by C. militaris obtained by adding different conditions of corncob biochar in solid media. Valuable results were obtained. However, this study looks more like a preliminary exploratory test than a systematic study.
Introduction would be better modified to explain the importance and innovation of this research logically and clearly.
How about the results of solid media contained 15 g of rice berry mixed with biochar over 10g?
The lack of detailed mechanism analysis in the results and discussion leads to the low persuasiveness of the paper.
The full text is relatively rough, and the sentence on line 158 is a bit repetitive. It is best to modify the English grammar. In addition, there are details errors in the text, such as Line 77, Line 128 and Line 154 and so on.
Author Response
Response to the reviewer comments
#Reviewer 1
Comment 1: This study investigated effects of medium components on cordycepin and adenosine production by C. militaris obtained by adding different conditions of corncob biochar in solid media. Valuable results were obtained. However, this study looks more like a preliminary exploratory test than a systematic study.
Introduction would be better modified to explain the importance and innovation of this research logically and clearly.
Answer: We are focusing on using Biochar which is contain a small molecules of carbon source in cultivation medium for Cordyceps to enhance the dry weight, Cordycepin yield and Adenisine yield. Additionally, we also focus on the morphology of biochar. The pore generates space for the mycelium to gather nutrients in solid medium and provides a carbon source for C. militaris which is the important nutrient for the fungal growth. This is the combination between the optimization of cultivation medium and the morphology of biochar that can enhance mycelium growth and provide carbon source for C.militaris as well as this is new finding to apply agricultural waste in cultivation. The introduction provide the series of Cordyceps cultivation, the factors and align with our finding. This is the first time to use biochar in Cordyceps cultivation. Thus, this is an importance of our study and new to science.
Comment 2: How about the results of solid media contained 15 g of rice berry mixed with biochar over 10g?
Answer: As biochar has very fine particle, 10g biochar is already turn the media to black and it effect to the fruiting body shape and the attachment of the fruiting body with the media as well as harvest of Cordyceps. Therefore the maximum of our experiments is 10g of biochar.
Comment 3: The lack of detailed mechanism analysis in the results and discussion leads to the low persuasiveness of the paper.
The full text is relatively rough, and the sentence on line 158 is a bit repetitive. It is best to modify the English grammar. In addition, there are details errors in the text, such as Line 77, Line 128 and Line 154 and so on.
Answer: The sentence has been modified. And error in text have been corrected.
Author Response File: 
Author Response.pdf
Reviewer 2 Report
1. Keywords: adenosine, agricultural residue, cultivation, cordycepin, SEM. I don't think SEM is a keyword.
2. It is recommended to divide the Introduction section one paragraph into 2 or 3 paragraphs.
3. Abbreviations should be noted at their first occurrence. Note Line 96 cordycepin yield (CY) , line 58 cordycepin yield. Please check other abbreviations.
4. Line 84-86, Regarding the preparation of biochar, it is recommended to add the reference "Investigations on the pyrolysis of microalgal-bacterial granular sludge: Products, kinetics, and potential mechanisms".
5. Line 136, The energy-dispersive X-ray spectroscopy (EDS) allowed for targeted analysis of sample surfaces (EDS, Oxford/Ultim Max 40 mm2) in order to determine the content of C and O in the mycelium of C. militaris. The energy-dispersive X-ray spectroscopy (EDS) only detects elements in a small part, and cannot replace the whole. EDS is used to indicate that the content of C and O is incorrect. The content of CHNS/O needs to be detected by elemental analysis.
6. In the manuscript, some places are written as 10g, 50days, and some places are 10g, 50 days, please unify the format, and pay attention to the spaces.
7. In the experimental design stage, the total amount of rice berry+biochar should be the same, and then the ratio of rice berry and biochar should be changed. For example, 1g rice berry+10 biochar, 5g rice berry+6 biochar. But I didn't see that you designed it this way. Please explain the reason for your design.
Author Response
#Reviewer 2
- Keywords: adenosine, agricultural residue, cultivation, cordycepin, SEM. I don't think SEM is a keyword.
Answer: remove SEM
- It is recommended to divide the Introduction section one paragraph into 2 or 3 paragraphs.
Answer: Split the introduction to 3 paragraphs, guess this was merge when we did the formatting.
- Abbreviations should be noted at their first occurrence. Note Line 96 cordycepin yield (CY) , line 58 cordycepin yield. Please check other abbreviations.
Answer: Corrected
- Line 84-86, Regarding the preparation of biochar, it is recommended to add the reference "Investigations on the pyrolysis of microalgal-bacterial granular sludge: Products, kinetics, and potential mechanisms".
Answer: The reference "Investigations on the pyrolysis of microalgal-bacterial granular sludge: Products, kinetics, and potential mechanisms" was added in the revised manuscript.
- Line 136, The energy-dispersive X-ray spectroscopy (EDS) allowed for targeted analysis of sample surfaces (EDS, Oxford/Ultim Max 40 mm2) in order to determine the content of C and O in the mycelium of C. militaris. The energy-dispersive X-ray spectroscopy (EDS) only detects elements in a small part, and cannot replace the whole. EDS is used to indicate that the content of C and O is incorrect. The content of CHNS/O needs to be detected by elemental analysis.
Answer: Because it was difficult to separate the mycelium of cordyceps from the medium (including biochar), therefore the SEM-EDS technique was used to identify the point of mycelium and the element content.
- In the manuscript, some places are written as 10g, 50days, and some places are 10g, 50 days, please unify the format, and pay attention to the spaces.
Answer: Corrected
- In the experimental design stage, the total amount of rice berry+biochar should be the same, and then the ratio of rice berry and biochar should be changed. For example, 1g rice berry+10 biochar, 5g rice berry+6 biochar. But I didn't see that you designed it this way. Please explain the reason for your design.
Answer: The experiment designed based on the optimization of Biochar and use rice berry as media base. Varies of concentration of biochar as we would like to apply the corncarb biochar which is enrich C source for Cordyceps cultivation medium plays an important role in the growth and the production of bioactive compounds. Various compounds can be used as a carbon and energy source by fungi. Carbon compounds range from simple small molecules like sugars, organic acids, and alcohols to proteins, polysaccharides, and lipids. Thus, using biochar which is contain a small molecules of carbon source for Cordyceps growing can enhance the dry fruiting body weight (DFW) as long as with cordycepin yield (CY), and adeno-sine yield (AY). Therefore, our study aimed to optimize the conditions for culturing C. militaris and establish bioactive compounds produced by C. militaris cultivates from biochar.
Author Response File: Author Response.pdf
Reviewer 3 Report
Find the enclosed file
Comments for author File: Comments.pdf
Author Response
#Reviewer 3
- Line 42: Provide some examples of physiologically active substances.
Answer: Some examples of physiologically active substances are added.
- Line 47: Citation No. 8 does not contain the study about the culture requirements for metabolites production. It deals with the in vitro antitumor activity of cordycepin. Give some (2-3) latest references that support the statement.
Answer: Reference No.8 is deleted and Add 3 (No.8, 9, 10) new references.
- Line 58-60: Cite the latest research articles for raw materials mentioned with production level cordycepin.
Answer: The references are cited.
- Line 64-65: Write the name of medium composition and culture conditions with relevant citations.
Answer: Revised.
- Line 67: Which many conditions? and
- Line 67-68: Incomplete sentence. Need to rewrite it.
Answer: Revised.
- Line 101: 'C. militaris' should be in italics. Revise the whole manuscript for such type of mistakes.
Answer: Revised.
- Line 103: Write 'C. militaris' after it appears first place in the manuscript. Check the whole manuscript for this inconstancy.
Answer: Checked and revised.
- Line 106: Provide the weight or percentage of the seed culture used during this experiment and other sets of the experiment. Is the same concentration of seed culture used throughout the study?
Answer: Yes, we use 50 ml of seed culture for all condition.
- Line 127: What for UV wavelength of 260 nm used?? Clarify.
Answer: Answer: The standard of cordycepin and adenosine were purchased from PhytoLab GmbH & Co. (Lot No. 82505) and Sigma Chemical Corporation (Lot No. WXBC586V), which showed the maximum wavelength at 259 nm. Therefore, the experiment selected used 260 nm for Cordycepin and Adenocien analysis. So we add the reference [30] and “Standards of cordycepin and adenosine were purchased from PhytoLab GmbH & Co. (Vestenbergsgreuth, Germany) and Sigma Chemical Corporation (St. Louis, MO, USA), respectively.” online 129.12.
- Line 150-154: What is the significance of the present study? The DFW of present study (i.e. 3.685g) is relatively lower than the other mentioned study.
Answer: However, our study report about CY and AY contents while not present in Song et al., (2010) and either in Kim et al., (2010)
And the significant is the new finding that we use biochar as the component of cultivation medium which will object to serve the carbon source.
This is the first report to use agricultural waste in term of biochar which is induced the cordycepin yield of Cordycpes.
- Line 175: What is the percentage or fold increment in cordycepin production?
Answer: As a result, found cordycepin increased by 100% when adding 10 g biochar in rich berry media which is the maximum of our experiment. However, we found that biochar does not affect adenosine production
- Line 200: Citation No [26]…….Use the proper way of citing. The sentence needs to be check for grammatical errors.
Answer: Revised.
- Line 204: Show the comparison of the titers of cordycepin production of the present study with the other latest studies.
Answer: Revised.
- Add the discussion of SEM results and analysis in the discussion section.
Answer: Revised. The discussion of SEM results were discussed in discussion section.
- Use proper citation format in table 3.
Answer: Revised and rearranged.
- The latest studies (last five years) need to be incorporated in table 3. Why has no study after 2016 been included in table 3?
Answer: Revised. Already added new references to table 3.
- Why One factor at a time approach has been used in the present study to obtain the optimum conditions. The use of statistical optimization approaches such as response surface methodology can be used.
Answer: Revised.
- The discussion section needs to be revised, especially by comparing the present study's findings with other latest studies to emphasize the significance of this study.
Answer: Revised.
- Must include last 3 years of study in the discussion.
Answer: Revised.
- Several references doi is missing such as ref no. 3, 5, 8, 9, 16, 21, 22, 23, 28. Add them.
Answer: All doi or the web link were added.
Author Response File: 
Author Response.docx
Round 2
Reviewer 1 Report
Accept in present form.
Reviewer 2 Report
accept
