Addition of Conductive Materials to Support Syntrophic Microorganisms in Anaerobic Digestion
, Elisa Pianta 2, Antoine Buetti 2, Federica Mauri 2, Matteo Tanadini 3 and Pamela Principi 1,*Round 1
Reviewer 1 Report
The manuscript fermentation-1813065 deals with the effect of conductive materials on the biogas production of ethanol batch digesters. The paper is of scientific interest. I may suggest some minor revision before it can be accepted for publication.
Please find my major comments on this work as follows:
1. The role of syntropic microorganisms in anaerobic digesters is not clearly described. The authors are suggested to give more detailed bibliographic analysis concerning this aspect. The previous studies about the tested conductive materials should be reported as well.
2. The characteristics of inoculum (TS, VS…) are not available. The substrate/inoculum ratio for BMP tests should be reported.
3. Section 2.4, why are the concentrations of conductive materials set at theses values?
4. Please further comment on Figure 1 and indicating the possible zones where the materials are found.
5. Figure 2, was the endogenic production of inoculum removed from the gas production?
6. Lines 237-241, what is the difference between the first trial and this second trial? Why did the second trial allow to understand if and how the material characteristics had an influence? Please specify it.
7. The quality of the figures should be improved.
8. The sole carbon souce is ethanol, why do you have the two peaks with the first one attributed as readily degradable OM ?
9. Lines 375-380, what are the roles of theses microorganisms with high correlation in the anaerobic digestion?
Author Response
here below the answers to the points arisen by reviewer # 1.
- The role of syntropic microorganisms in anaerobic digesters is not clearly described. The authors are suggested to give more detailed bibliographic analysis concerning this aspect. The previous studies about the tested conductive materials should be reported as well.
Answer: The introduction has been restructured evidencing the syntrophy and its occurrence in AD processes.
In particular, the following paragraph has been added: (lines 67-76) “Factors facilitating interspecies electron transfer such as redox-active compounds and conductive materials, improve the syntrophy between bacteria and methanogenic archaea needed to overcome the thermodynamic limits. Recent studies have suggested that conductive iron oxide minerals can facilitate syntrophic metabolism of the methanogenic degradation of organic matter such as ethanol, propionate and butyrate in natural and engineered microbial ecosystems [8]. Different conductive carbon materials have been proved able to support and facilitate DIET process in lab scale with pure culture microorganisms [9][10]. They include graphite particles [11], granular activated carbon [12], biochar [13], and carbon cloth [14]. Cruz Viggi et al. [15] tested the addition of microparticulated magnetite (Fe3O4) in a real anaerobic digestion process.” And later (lines 81-87) “Although recent research focuses on DIET-related microorganisms to accelerate and stabilize the biochemical reactions involved in the methane production (Martins et al., 2018, Mostafa et al., 2020), there is still the need to relate the effect of the material addition with conductivity values and physical properties as the material characteristics (dimension, surface) may affect the DIET efficiency [18]. A deeper knowledge on the microbial community dynamics is fundamental to exploit DIET-associated syntrophy in real scale application to increase efficiency.”
- The characteristics of inoculum (TS, VS…) are not available. The substrate/inoculum ratio for BMP tests should be reported.
Answer: The missing inoculum characteristics have been added in line 112-114 “The digestate sampled for inoculum had a Total Solid content (TS) of 23.6g/L with a 60.4% of organic matter measured as VS. The volatile solids ratio between inoculum and feeding was 1:1.”
- Section 2.4, why are the concentrations of conductive materials set at theses values?
Answer: Different materials with a wide range of concentrations were preliminary tested. The ones giving interesting results were chosen to be further tested as it is written in line 114.
- Please further comment on Figure 1 and indicating the possible zones where the materials are found.
Answer: A more detailed description of the figure has been added. Lines 208-213 “At lower magnification (figure 1 upper line from left to right) zeolite reveals particles in the range of a few microns with a high variance, biochar presents 100-200 µm mate-rial clusters, graphene shows agglomerate structure and PAC has an uniform distribu-tion; zooming in at 4500x (bottom line figure 1) all the materials have very wide parti-cle morphology with graphene showing sharp structures.”
- Figure 2, was the endogenic production of inoculum removed from the gas production?
Answer: As inoculum we chose microbial consortia from a real scale digester fed with WWTP sludge from municipal wastewater. To consider the variability in the microbial community structure and function, we decided to set independent controls for each tests. The biogas production of the inoculum is considered in the experiments by running parallel controls set with the same inoculum. In figure 2 two sets of tests are reported and each has its own control. The paragraph in line 238-242 has been modified to make more undersatandable this concept “To consider the effect of the microbial composition in the inoculum as variable and the endogenous biogas production, PAC and biochar treatment were set with the very same inoculum as CTRL-PB, while graphene and zeolite have the same inoculum as CTRL-GZ. Graphene reached the plateau after 150 hours, while zeolite and CTRL-GZ around 70 hours. PAC, biochar and their control show a similar trend.”
- Lines 237-241, what is the difference between the first trial and this second trial? Why did the second trial allow to understand if and how the material characteristics had an influence? Please specify it.
Answer: there is no difference between first and second trial that was run to confirm the results and allow for molecular analysis of the communities. The sentence in line 262-264 has been modified accordingly “The test settings were the same as the previous experiments and a reactor with no material supplement was set as control. In this second run samples for microbial community analysis were collected at given times (S in the figure 4).”
- The quality of the figures should be improved.
Answer: done.
- The sole carbon souce is ethanol, why do you have the two peaks with the first one attributed as readily degradable OM ?
Answer: it is true that we used ethanol as feeding to have data comparable with other research, however, by choosing as inoculum real digestate we are adding other organics that could be metabolized. The sentence was modified to make it more understandable in lines 362-366 “Considering that the reactors were run in batch mode, the first peak represents the conversion of readily degradable organic matter present in the ethanol-feeding, while in the second one the microbial activity is supported by the conversion of the remaining organics that are anyway present in the digestate used as inoculum.”
- Lines 375-380, what are the roles of theses microorganisms with high correlation in the anaerobic digestion?
Answer: In lines 413-421 we added a paragraph proposing a role of the correlated taxa found in the graphene-added reactors : “Ca. Methanofastidiosum uses the methylotrophic pathway to produce methane [10] and resulted the dominant archaea in the inoculum and during the process in all the tests. Considering then that a positive correlation was found for Geobacter sp. and ca. Methanofastidiosum just in the graphene added tests, we can state that graphene addition stimulates a syntrophic methylotrophic metabolism between Geobacter sp. and ca. Methanofastidiosum. Moreover as Geobacter sp. is active in electron transfer syntrophy [10][31] we conclude that in our tests the presence of PAC and graphene promotes a diverse syntrophic microbial community resulting in higher biogas production and in particular graphene promotes a interspecies electron transfer between for Geobacter sp. and ca. Methanofastidiosum.”
Reviewer 2 Report
Abstract needs rephrasing for more clear understanding
-introduction of the manuscript require the recent references and literature
-authors needs to write the research gaps and how this findings going to contribute better than earlier researchers observations
-in abstract authors needs to give more information
-mathematical annotations could be written in proper format many mistakes in the present version
-the axis of the figures not written in a clear font –
Author Response
The answer to the points arisen by reviewer 2 are hereinafter reported.
Reviwer# 2
Abstract needs rephrasing for more clear understanding
Answer: the abstract has been rewritten to include more information on the results presented.
-introduction of the manuscript require the recent references and literature
Answer: in the introduction we added more recent references and relevant to the conductive material addition
-authors needs to write the research gaps and how this findings going to contribute better than earlier researchers observations
Answer: A paragraph detailing the contribution of the present paper to the topic of conductive material to improve biogas production efficiency has been added in the abstract “This paper contributes to the understanding of the DIET-related microbial community dynamic in presence of graphene and PAC, that could be exploited to optimize biogas and methane production in real scale applications.”, introduction (line 85-87) and conclusions (lines 435-439).
-in abstract authors needs to give more information
Answer: see previous point
-mathematical annotations could be written in proper format many mistakes in the present version
Answer: we changed the equation and fixed typos with the equation editor
-the axis of the figures not written in a clear font –
Answer: the figure have been included in the manuscript with higher fonts and better quality
