Potential of Cation Exchange Resin as a Carrier for Anaerobic Consortia in Biohydrogen Fermentation
Round 1
Reviewer 1 Report
Comments and Suggestions for AuthorsThe manuscript presents an interesting comparison between two types of cation exchange resins to be used as carriers for anaerobic consortia in biohydrogen fermentation.
The work is very interesting, with relevant applicative aspects.
The manuscript is well written in every part, however I found two relevant issues that must be addressed in order to make this work publishable.
A first issue was found in Line 172, concerning the analysis of Illumina sequencing data. The link https://www.majorbio.com send me to a website that I cannot read, because it does not use an international alphabet. Since a high number of people within the scientific community does not have access to this information, I would recommend providing in the manuscript a description of the pipeline that was used for the analysis, to make the experiment fully reproducible.
The second issue concerns the fact that the present data completely miss any statistical analysis. In order to make any inference from the results, a statistical analysis is required, at list at the descriptive level (average, standard deviation).
Author Response
Comments 1: A first issue was found in Line 172, concerning the analysis of Illumina sequencing data. The link https://www.majorbio.com send me to a website that I cannot read, because it does not use an international alphabet. Since a high number of people within the scientific community does not have access to this information, I would recommend providing in the manuscript a description of the pipeline that was used for the analysis, to make the experiment fully reproducible.
Response 1: Thank you for pointing this out. Therefore, we have added the description of the pipeline that was used for the analysis. Please see the Pages 4-5, lines 171-178, we have replaced “…The high-quality sequences were de-noised, and data analysis was performed on a Majorbio Cloud platform (https://cloud.majorbio.com).” with “…The high-quality sequences were de-noised, and denoised sequences are usually called amplicon sequence variants (ASVs). Taxonomic assignment of ASVs was performed using the Naive bayes consensus taxonomy classifier implemented in Qiime2 and the SILVA 16S rRNA database (v138). Bioinformatic analysis was performed using the Majorbio Cloud platform (https://cloud.majorbio.com). Based on the ASVs information, rarefaction curves and alpha diversity indices including observed ASVs, Chao1 rich-ness, Shannon index and Good’s coverage were calculated with Mothur v1.30.1."
Comments 2: The second issue concerns the fact that the present data completely miss any statistical analysis. In order to make any inference from the results, a statistical analysis is required, at list at the descriptive level (average, standard deviation).
Response 2: Thank you for pointing this out. According to the results of three parallel samples, we have conducted the corresponding statistical analysis and the standard deviation values were obtained. Please see the Figures 1~3 and Tables 3 and 4 in the revised manuscript, error bars have been added to enhance the statistical significance.
Reviewer 2 Report
Comments and Suggestions for AuthorsThe aim of the study was to investigate cation exchange resins as supports for acidogenic biomass. In general, the study does not seem to have been carried out systematically, e.g. the conditions of fermentation are not clearly described, the number of replicates, etc. From my point of view, after reading the paper, I find it difficult to consider it as a novel study with a significant contribution to the state of the art, so I do not recommend it for publication.
Author Response
Comment 1: The aim of the study was to investigate cation exchange resins as supports for acidogenic biomass. In general, the study does not seem to have been carried out systematically, e.g. the conditions of fermentation are not clearly described, the number of replicates, etc. From my point of view, after reading the paper, I find it difficult to consider it as a novel study with a significant contribution to the state of the art, so I do not recommend it for publication.
Response 1: Thank you for your comment. Firstly, we have introduced the fermentation conditions in detail in Section 2.2. Please see the Page 3, lines 98-111, “The 2-day fermentation experiment was conducted using eight 500 mL serum bottles, each with a working volume of 400 mL. The substrate and inoculum were added to six bottles at a VS ratio of 5:1. Four bottles, labelled as experimental group-1 (EG-1), contained 4 g CER-1/g VS; and the remaining four bottles, labelled as experimental group-2 (EG-2), contained 4 g CER-2/g VS. The CER dosage was based on our prior study [8]. The pH of the mixture in each bottle was adjusted to approximately 6.0 using 1 M NaOH or HCl solutions. Each bottle was flushed with N2 for 3–5 min to purge oxygen, sealed promptly, and placed in a water bath at 37.0 ± 1.0 ℃ with mechanical stirring at 120 rpm. After 2-day fermentation, the CER was separated from the fermented sludge by sieving the mixture through a screen mesh (ø 20 × 4.5 cm, 50 mesh). The separated CER (S-CER) from one bottle in each group was used for characterisation, while those from the other three bottles in each group were used for the subsequent fermentation experiments, as described in Section 2.4. The separated fermented sludge was used for microbial community analysis. The S-CER from this short-term fermentation was labelled as SS-CER.”; and the Page 3, lines 112-117, “The 28-day fermentation experiment was also conducted using eight 500 mL se-rum bottles, each with a working volume of 400 mL. The inoculum and CER were mixed directly, without substrates. The other procedures were the same as those in the 2-day fermentation experiment, except that the pH of the mixture in each bottle was adjusted to approximately 7.0 using 1 M NaOH or HCl solutions. The S-CER from this long-term anaerobic fermentation was labelled as LS-CER.” Secondly, the experiments were constructed in triplicates. Please see the Page 3, lines 100-102, “…Four bottles, labelled as experimental group-1 (EG-1), contained 4 g CER-1/g VS; and the remaining four bottles, labelled as experimental group-2 (EG-2), contained 4 g CER-2/g VS…”; the Page 3, lines 112-113, “The 28-day fermentation experiment was also conducted using eight 500 mL se-rum bottles, each with a working volume of 400 mL…”. Additionally, according to the results of three parallel samples, we have conducted the corresponding statistical analysis and the standard deviation values were obtained. Please see the Figures 1~3 and Tables 3 and 4 in the revised manuscript, error bars have been added to enhance the statistical significance. Thirdly, as we summarized in the introduction, most studies about CER-enhanced anaerobic fermentation of sludge focused on the influence of CER-mediated ion exchange reactions on the sludge fermentation performance, but the potential of as a biocarrier for bacterial adhesion and growth was overlooked in these studies, and also, these studies have not characterised CER post-fermentation or determined whether anaerobic consortia can adhere to the CER surface. This study was first to explore the ability of CER to serve as a carrier for anaerobic consortia during sludge fermentation, which is also the novelty of this study.
Reviewer 3 Report
Comments and Suggestions for Authors
1-The authors must mention the type of sludge collected (activated or any other type). Sometimes is confusing whether the the sludge was used as inoculum or substrate.
2-Please report the current as current density (mA/cm2) throughout the text and in figures.
3-The error bars are not provided in the figures. Did the experiments were run in triplicates? statistical analysis needs to be performed.
4-In Figures 4 a and b, up to 60% of the microbial populations are shown. What was the rest of the community? please normalise these figures to 100% as shown for Fig 4c.
Author Response
Comment 1: The authors must mention the type of sludge collected (activated or any other type). Sometimes is confusing whether the sludge was used as inoculum or substrate.
Response 1: Thank you for your comment. Please see the Page 2, lines 82-83, we have modified the sentences and replaced “Sludge was collected from a wastewater treatment plant in Shanghai, China…” with “Sewage sludge, as substrate, was collected from a wastewater treatment plant in Shanghai, China…”
Comment 2: Please report the current as current density (mA/cm2) throughout the text and in figures
Response 2: Agree. Therefore, we have replaced current as current density. Please see the Figure 2(a) in the revised manuscript, we have made the modification.
Comment 3: The error bars are not provided in the figures. Did the experiments were run in triplicates? statistical analysis needs to be performed.
Response 3: Thank you for pointing this out. Firstly, the experiments were constructed in triplicates. Please see the Page 3, lines 100-102, “…Four bottles, labelled as experimental group-1 (EG-1), contained 4 g CER-1/g VS; and the remaining four bottles, labelled as experimental group-2 (EG-2), contained 4 g CER-2/g VS…”; the Page 3, lines 112-113, “The 28-day fermentation experiment was also conducted using eight 500 mL se-rum bottles, each with a working volume of 400 mL…”. Secondly, according to the results of three parallel samples, we have conducted the corresponding statistical analysis and the standard deviation values were obtained. Please see the Figures 1~3 and Tables 3 and 4 in the revised manuscript, error bars have been added to enhance the statistical significance.
Comment 4: In Figures 4 a and b, up to 60% of the microbial populations are shown. What was the rest of the community? please normalise these figures to 100% as shown for Fig 4c.
Response 4: We agree with this comment. Therefore, please see the Figure 4 in the revised manuscript, we have modified this figure.
Round 2
Reviewer 2 Report
Comments and Suggestions for AuthorsAfter reading the study, I believe that it should provide sufficient details about the fermentations performed and present the results, not just at one point of the fermentation, but throughout the fermentation kinetics. Therefore, I do not consider the current work acceptable for publication in its present form.
