Rapid Two Stage Anaerobic Digestion of Nejayote through Microaeration and Direct Interspecies Electron Transfer
Round 1
Reviewer 1 Report
The authors have demonstrated the utility of including both micro aeration and GAC to improved methanogenesis from nejayote.
Overall this is good research. The ms. will benefit from some questions about specific details, but will require extensive revision to improve the scientific English.
line 121. Provide the exact source and catalog number for the GAC used. This is quite variable from source to source.
lines 341-344. Check the species of Peptoclostridium that matched and were used for sequence identification. At least one database has incorrectly named Clostridiodes difficile as Peptoclostridium difficile. Also, this genus is best known for metabolizing amino acids, so it is not perfectly clear how it is participating in this process.
Remaining comments will focus on improvement of the presentation/scientific English. These will not be exhaustive. The authors must review their ms. word by word and conduct a final revision of the ms.
lines 2, 58 and elsewhere. Use the singular "two stage".
line 20 Change to "biofilm, though this....".
lines 35, 88, 104, 276, 312 and elsewhere. Remove the excessive commas.
line 38 Change to "process, which can lead to low yields in methanols production....". This is also an example of excessive use of "the", which will be addressed below.
lines 40, 43, 53, 102, 124, 127, 162, 165, 278, 288 and elsewhere. Omit the excessive "the"s. Similarly, there are places where "the" should be replaced with "a", such as line 277.
lines 45 and 241. Use separate sentences here rather than a ";".
line 48 Change to "however the overall methanogenic reaction....".
line 49 Change to "value, making this....".
line 64 Omit "in an ultra-freezer".
line 67 What is "deep soil"?
lines 77-78 and 83. These statement are repetitive.
lines 78 and 83. Just say "was used".
line 87 Change to "maintained with a circulating water bath.".
line 100. 7.5 cm PVC rings seem large for this size system.
line 102 "packing material".
lines 122 and 124. Omit "cloth".
lines 152 153. Start with "DNA extraction", then end with the phrase "at the end....". Start with the subject of the verb and verb whenever possible. Avoid starting a sentence with a phrase.
line 198. Was this hydrogen captured for subsequent methanogenesis?
lines 216-217. Unsure of the meaning of this sentence.
lines 241, 287 and 312. Replaced "experimentation" with "experiment".
Author Response
RESPONSE TO REVIEWERS
Manuscript ID: processes-996488
Type of manuscript: Article
Title: RAPID TWO STAGES ANAEROBIC DIGESTION OF NEJAYOTE THROUGH MICROAERATION AND DIRECT INTERSPECIES ELECTRON TRANSFER
Authors: David Valero, Carlos Rico, Raul Tapia-Tussell and Liliana Alzate-Gaviria* (corresponding author)
RESPONSE TO COMMENTS FROM REVIEWER 1
The authors have demonstrated the utility of including both micro aeration and GAC to improved methanogenesis from nejayote. Overall this is good research. The ms. will benefit from some questions about specific details, but will require extensive revision to improve the scientific English.
- line 121. Provide the exact source and catalog number for the GAC used. This is quite variable from source to source.
Response: Carbotecnia-25 Kg-Activated carbon from bituminous coal-granulometry 12 × 40.
- lines 341-344. Check the species of Peptoclostridium that matched and were used for sequence identification. At least one database has incorrectly named Clostridiodes difficile as Peptoclostridium difficile. Also, this genus is best known for metabolizing amino acids, so it is not perfectly clear how it is participating in this process.
Response: In line 341-344 it is clarified that Peptoclostirium and Clostridium are among the bacteria with the highest abundance in the carbon biofilm and play an important role in the transfer of electrons. From 2014, the DIET between exoelectrogenic bacteria and electrotrophic methanogens was evidenced through pili in a Geobacter-Methanosaeta co-culture [12,13]. Over the past six years, through the microorganism identification of biofilms enriched in conductive materials, new exoelectrogenic bacteria have been discovered, such as Thauera, Sporanaerobacter, Enterococcus, Pseudomonas, Anaerolinaceae, Bacteroides, Streptococcus, Syntrophomonas, Sulfurospirillum, Caloramator, Tepidoanaerobacter, Coprothermobacter, Clostridium senso stricto, Peptococcaceae, and Bacillaceae, also new electrotrophic methanogen genera have been found able to participate in DIET, such as Methanosarcina, Methanobacterium, Methanolinea, Methanothrix, Methanoregula and Methanospirillum [14,18,20]. However, the number of bacteria that are capable of participating in DIET through conductive materials is still unknown [14.21].
Remaining comments will focus on improvement of the presentation/scientific English. These will not be exhaustive. The authors must review their ms. word by word and conduct a final revision of the ms.
- lines 2, 58 and elsewhere. Use the singular "two stage".
Response: Modification was made.
- line 20 Change to "biofilm, though this....".
Response: Change was made.
- lines 35, 88, 104, 276, 312 and elsewhere. Remove the excessive commas.
Response: Commas were removed.
- line 38 Change to "process, which can lead to low yields in methanols production....". This is also an example of excessive use of "the", which will be addressed below. lines 40, 43, 53, 102, 124, 127, 162, 165, 278, 288 and elsewhere. Omit the excessive "the"s. Similarly, there are places where "the" should be replaced with "a", such as line 277.
Response: Excessive use of “the”s were deleted and text was rewritten.
- lines 45 and 241. Use separate sentences here rather than a ";".
Response: Modification was made.
- line 48 Change to "however the overall methanogenic reaction....".
Response: Change was made.
- line 49 Change to "value, making this....".
Response: Change was made.
- line 64 Omit "in an ultra-freezer".
Response: Change was made.
- line 67 What is "deep soil"?
Response: The statement was changed as follows: which consisted of soil (30 cm deep) 30 g L-1…
- lines 77-78 and 83. These statement are repetitive.
Response: 77-78 lines deleted.
- lines 78 and 83. Just say "was used".
Response: Change was made.
- line 87 Change to "maintained with a circulating water bath.".
Response: Change was made.
- line 100. 7.5 cm PVC rings seem large for this size system.
Response: 7.5 cm was a mistake, correct measure was 7.5 mm.
- line 102 "packing material".
Response: Modification was made.
- lines 122 and 124. Omit "cloth".
Response: “cloth” was omit.
- lines 152 153. Start with "DNA extraction", then end with the phrase "at the end....". Start with the subject of the verb and verb whenever possible. Avoid starting a sentence with a phrase.
Response: Modification was made.
- line 198. Was this hydrogen captured for subsequent methanogenesis?
Response: Hydrogen was not captured for subsequent methanogenesis.
- lines 216-217. Unsure of the meaning of this sentence.
Response: The sentence was deleted.
- lines 241, 287 and 312. Replaced "experimentation" with "experiment".
Response: Modifications were made.
Author Response File: 
Author Response.pdf
Reviewer 2 Report
In the present manuscript a two stage anaerobic digestion system was employed to treat nejayote wastewater of increased pH. Microaeration was applied in order to increase hydrolytic activity and aid VFAs production. The COD removal was moderate and the methane yield acceptable. The language is of sufficient quality. Moreover, the quality of the figures and tables is satisfactory.
There is no reference in the introduction regarding the microbiology of such wastewater. The authors should consider including a paragraph on microorganisms.
The physiochemical analysis of the raw material is poor. Usually analyses are performed in triplicate, especially in cases where the COD concentration has such a significant standard deviation. There is no VFAs measurement in the wastewater and no estimation on the sulphate content. Moreover the phenol concentration was not measured, as well as protein and sugars (soluble and non-soluble) content [Nejayote contains large insoluble polymers (cellulose, hemicellulose and lignin) which are difficult to be biodegraded]. Phosphorus and nitrogen content is required to judge whether their concentration satisfies growth needs of the microorganisms performing the biological processes.
Could the authors also clarify if the wastewater was introduced in the hydrolytic reactor without any pH adjustment?
Genus of Peproclostridium consists of bacteria, which are pathogenic for humans. How do you explain the fact that these bacteria covered a significant part of the relative abundance of the examined samples?
Amonobacterium are characterized as amino acid degraders and seem also capable of performing sulphate reduction. Moreover, they constitute a dominant genus in all the reactor assemblies the authors applied. However, their role and presence in the described processes is overlooked. Desulfotomaculum covers 4.3% of the relative abundance, are sulphate reducers, producing sulfide as well as acetate but still their role is not discussed in the manuscript.
The operational period for the anaerobic reactor seems limited, considering the time requirements for the microorganisms to grow under these conditions. In many cases HRT of 40 or 60 days is applied in order to increase methane yield. Could the authors include information on the HRT they applied?
Author Response
RESPONSE TO REVIEWERS
Manuscript ID: processes-996488
Type of manuscript: Article
Title: RAPID TWO STAGES ANAEROBIC DIGESTION OF NEJAYOTE THROUGH MICROAERATION AND DIRECT INTERSPECIES ELECTRON TRANSFER
Authors: David Valero, Carlos Rico, Raul Tapia-Tussell and Liliana Alzate-Gaviria* (corresponding author)
RESPONSE TO COMMENTS FROM REVIEWER 2
In the present manuscript a two stage anaerobic digestion system was employed to treat nejayote wastewater of increased pH. Microaeration was applied in order to increase hydrolytic activity and aid VFAs production. The COD removal was moderate and the methane yield acceptable. The language is of sufficient quality. Moreover, the quality of the figures and tables is satisfactory.
- There is no reference in the introduction regarding the microbiology of such wastewater. The authors should consider including a paragraph on microorganisms.
Response: From 2014, the DIET between exoelectrogenic bacteria and electrotrophic methanogens was evidenced through pili in a Geobacter-Methanosaeta co-culture [12,13]. Over the past six years, through the microorganism identification of biofilms enriched in conductive materials, new exoelectrogenic bacteria have been discovered, such as Thauera, Sporanaerobacter, Enterococcus, Pseudomonas, Anaerolinaceae, Bacteroides, Streptococcus, Syntrophomonas, Sulfurospirillum, Caloramator, Tepidoanaerobacter, Coprothermobacter, Clostridium senso stricto, Peptococcaceae, and Bacillaceae, also new electrotrophic methanogen genera have been found able to participate in DIET, such as Methanosarcina, Methanobacterium, Methanolinea, Methanothrix, Methanoregula and Methanospirillum [14,18,20]. However, the number of bacteria that are capable of participating in DIET through conductive materials is still unknown [14.21].
- The physiochemical analysis of the raw material is poor. Usually analyses are performed in triplicate, especially in cases where the COD concentration has such a significant standard deviation. There is no VFAs measurement in the wastewater and no estimation on the sulphate content. Moreover, the phenol concentration was not measured, as well as protein and sugars (soluble and non-soluble) content [Nejayote contains large insoluble polymers (cellulose, hemicellulose and lignin) which are difficult to be biodegraded]. Phosphorus and nitrogen content is required to judge whether their concentration satisfies growth needs of the microorganisms performing the biological processes.
Response: Analysis of the raw material was added, as follows:
Table 1. Nejayote characterization compared with other studies.
|
Parameter |
Results |
Other Studies [3,4] |
|
pH |
10.20 |
6.30–11.60 |
|
COD (mg L−1) |
15,433 ± 826 |
3,430–40,058 |
|
NH3-N (mg L−1) |
4.65 ± 0.05 |
2–4 |
|
TN (mg L−1) |
95.33 ± 3.7 |
209–428 |
|
SO4−2 (mg L−1) |
22.50 ± 2.5 |
13–14 |
|
PO4−3 (mg L−1) |
58.75 ± 1.2 |
7.6–1321 |
|
TA (mg CaCO3 L−1) |
1,799 ± 116 |
5,768 |
|
TSS (mg L−1) |
2,676 ± 512 |
1,810–8,340 |
|
TS % |
1.22 ± 0.01 |
0.34–2.5 |
|
VS % |
0.84 ± 0.01 |
0.24–1.55 |
|
VS/TS |
0.69 ± 0.01 |
0.7 |
total alkalinity (TA), total suspended solids (TSS), total nitrogen (TN), phosphates (PO4−3), ammonia nitrogen (NH3N), sulfate (SO4−2)
- Could the authors also clarify if the wastewater was introduced in the hydrolytic reactor without any pH adjustment?
Response: The wastewater was introduced inside hydrolytic reactor without any pH adjustment and was added in text.
- Genus of Peproclostridium consists of bacteria, which are pathogenic for humans. How do you explain the fact that these bacteria covered a significant part of the relative abundance of the examined samples?
Response: These bacteria can be found in the gastrointestinal tracts of both humans and animals, in our study, the inoculum used in both reactors was a mixed microbial consortium, which consisted of deep soil, dairy manure and swine manure. It is also important to point out that the raw material (Nejayote) used in this study has ideal properties for the growth of this type of bacteria (high level of alkalinity).
Reference: Pereira, F. L., Júnior, C. A. O., Silva, R. O., Dorella, F. A., Carvalho, A. F., Almeida, G. M., ... & Figueiredo, H. C. (2016). Complete genome sequence of Peptoclostridium difficile strain Z31. Gut pathogens, 8(1), 1-7.
- Aminobacterium are characterized as amino acid degraders and seem also capable of performing sulphate reduction. Moreover, they constitute a dominant genus in all the reactor assemblies the authors applied. However, their role and presence in the described processes is overlooked. Desulfotomaculum covers 4.3% of the relative abundance, are sulphate reducers, producing sulfide as well as acetate but still their role is not discussed in the manuscript.
Response: The following text was added to the article:
Aminobacterium are capable of degrading amino acids to VFAs. Aminobacterium colombiense and Clostridium sporogenes do not contain FDH (formate dehydrogenase) genes, which means they can only operate via H2 electron transfer in syntrophic metabolism. Some other substances besides H2 and formate have been reported as electron carriers in syntrophic metabolism [40,41,46].
This part is not added in the text, Aminobacterium are capable of degrading amino acids to VFAs. Short chain volatile fatty acids and alcohols are the biodegradable compounds in the syntrophic association between exoelectrogenic bacteria and electrotrophic methanogens. Aminobacterium colombiense, Clostridium sporogenes, and Thermanaerovibrio acidaminovorans do not contain FDH (formate dehydrogenase) genes, which means they can only operate via H2 electron transfer in syntrophic metabolism. Some other substances besides H2 and formate have been reported as electron carriers in syntrophic metabolism. During the co-culture of Geobacter sulfurreducens and Geobacter metallireducens, OmcS, a multiheme c-type cytochrome of G. sulfurreducens, can accept electrons from c-type cytochromes of G. metallireducens.
Reference: Leng, L., Yang, P., Singh, S., Zhuang, H., Xu, L., Chen, W. H., ... & Chu, W. (2018). A review on the bioenergetics of anaerobic microbial metabolism close to the thermodynamic limits and its implications for digestion applications. Bioresource technology, 247, 1095-1106.
- The operational period for the anaerobic reactor seems limited, considering the time requirements for the microorganisms to grow under these conditions. In many cases HRT of 40 or 60 days is applied in order to increase methane yield. Could the authors include information on the HRT they applied?
Response: The HRT was 6 days.
Author Response File: Author Response.pdf
Round 2
Reviewer 1 Report
A revised ms. which showed the utility of combining aerobic and anaerobic processing stages for the methanogenic biodegradation of nejayote was reviewed.
As noted previously, the research is of good quality and the work is of interest.
The presentation in the revision is significantly improved. Several suggestions for further improvement follow.
Suggested changes will be given as suggested new text.
line 20 "biofilm, though interestingly, Peptoclostridium....".
line 40 "low yields in methane production.".
line 56 "In order for DIET to occur,....".
lines 61-62 "six years, through identification of microorganisms in biofilms enriched....".
line 65 "Bacillaceae. Also, new electrotrophic....".
line 68 "bacteria capable of participating".
line 116 "polyethylene to avoid heat loss.".
lines 166, 272, 348 and 353 "experiment".
line 273 "control accumulated in this....". Use past tense rather than past perfect tense when possible.
line 308 "degradation performance was due to".
line 314 "The absence of acetic acid from the fourth day....".
line 353 "The total methane yield throughout the experiment was 55.5% higher....".
line 355 "materials were added....".
line 374 "three well defined....".
line 377 Is "than the previously mentioned also clustered...." better?
lines 388 and 390 "phyla".
line 394 "Pelotomaculum, were detected".
Ref.s 1, 14, 16, 27 and 37 are missing page numbers.
Ref.s 22, 30 and 32 Be consistent and complete in citation of books.
Ref.s 49, 56 and 57 need added italics of species names.
Author Response
RESPONSE TO COMMENTS FROM REVIEWER 1
A revised ms. which showed the utility of combining aerobic and anaerobic processing stages for the methanogenic biodegradation of nejayote was reviewed. As noted previously, the research is of good quality and the work is of interest. The presentation in the revision is significantly improved. Several suggestions for further improvement follow.
Suggested changes will be given as suggested new text.
- line 20 "biofilm, though interestingly, Peptoclostridium....".
Response: Modification was made.
- line 40 "low yields in methane production.".
Response: Change was made.
- line 56 "In order for DIET to occur,....".
Response: Change was made.
- lines 61-62 "six years, through identification of microorganisms in biofilms enriched....".
Response: Modification was made.
- line 65 "Bacillaceae. Also, new electrotrophic....".
Response: Modification was made.
- line 68 "bacteria capable of participating".
Response: Modification was made.
- line 116 "polyethylene to avoid heat loss.".
Response: Modification was made.
- lines 166, 272, 348 and 353 "experiment".
Response: Changes were made.
- line 273 "control accumulated in this....". Use past tense rather than past perfect tense when possible.
Response: Modification was made.
- line 308 "degradation performance was due to".
Response: Modification was made.
- line 314 "The absence of acetic acid from the fourth day....".
Response: Modification was made.
- line 353 "The total methane yield throughout the experiment was 55.5% higher....".
Response: Modification was made.
- line 355 "materials were added....".
Response: Modification was made.
- line 374 "three well defined....".
Response: Modification was made.
- line 377 Is "than the previously mentioned also clustered...." better?
Response: Yes, it is better.
- lines 388 and 390 "phyla".
Response: Changes were made.
- line 394 "Pelotomaculum, were detected".
Response: Modification was made.
- s 1, 14, 16, 27 and 37 are missing page numbers.
Response: 1, 27 and 37 page numbers were added. 14 and 16 are not missing page numbers.
- s 22, 30 and 32 Be consistent and complete in citation of books.
Response: 22 and 30 were completed in the citation as books. 32 is not a book.
- s 49, 56 and 57 need added italics of species names.
Response: Changes were made.
Reviewer 2 Report
Even-though authors kindly responded to all recommendations, the added part on microorganisms in the introduction seems roughly written. Bacterial species of the genera Thauera, Pseudomonas etc are not new bacteria. Methanosarcina are not new electrotrophic methanogenic genera. Introduction, as a part of the research, is an equally important section of the manuscript that requires attention.
Author Response
RESPONSE TO COMMENTS FROM REVIEWER 2
Even-though authors kindly responded to all recommendations, the added part on microorganisms in the introduction seems roughly written. Bacterial species of the genera Thauera, Pseudomonas etc are not new bacteria. Methanosarcina are not new electrotrophic methanogenic genera. Introduction, as a part of the research, is an equally important section of the manuscript that requires attention.
- Response: The word "new" was deleted, the reviewer is absolutely right. Likewise, some conductive materials, exoelectrogenic bacteria and electrotrophic methanogen were added to the introduction.
From 2014, the DIET between exoelectrogenic bacteria and electrotrophic methanogens was evidenced through pili in a Geobacter-Methanosaeta co-culture [12,13]. Six years, through identification of microorganisms in biofilms enriched in conductive materials (magnetite, granular activated carbon, biochar, graphene, iron oxide nanoparticles, hematite and akaganeite), exoelectrogenic bacteria have been discovered, such as Thauera, Sporanaerobacter, Enterococcus, Pseudomonas, Anaerolinaceae, Bacteroides, Streptococcus, Syntrophomonas, Sulfurospirillum, Caloramator, Tepidoanaerobacter, Coprothermobacter, Clostridium senso stricto, Peptococcaceae, Caloramator, Syntrophmonadaceae and Bacillaceae. Also electrotrophic methanogen genera have been found able to participate in DIET, such as Methanosarcina, Methanobacterium, Methanolinea, Methanothrix, Methanoregula, Methanimicrococcus, Methanosaeta and Methanospirillum [14,18,20]. However, the number of bacteria capable of participating in DIET through conductive materials is still unknown [14.21].
