Review Reports - Deeper Insights into the Effect of Humic Acid on Kitchen Waste Anaerobic Digestion: Enzyme Activities, Microbial Community Dynamics, and Key Metabolic Pathways

Round 1

Reviewer 1 Report

Anaerobic digestion is a processing technology that can efficiently recover resources and energy. Humic acid is a soluble microbial product generated during anaerobic digestion of kitchen waste. This study conducted batch experiments using kitchen waste as substrate, biogas slurry as inoculum, and six gradient concentrations of HA (HA/VS) as additive. By continuously monitoring indicates like cumulative methane production, reactive oxygen species (ROS) metabolism characteristics and microbial community structure and metabolic pathways during anaerobic digestion, the impact of HA on the entire process was investigated. The aforementioned study holds significant importance in promoting the efficient management and resource utilization of kitchen waste. The manuscript is within the scope of Fermentation and is interesting for readers. However, a few questions have been raised that should be answered prior to publication.

1. Line 125: Elaborate on the measurement steps of ROS in detail.

2. Line 256: Why does low concentration of humic acid promote the production of VFA? From Figure 2, it can be seen that low concentrations of humic acid still have a certain inhibitory effect on hydrolases.

3. Line 300: Change “possibly because of which may be due to” to “possibly because of”.

4. Line 375: Change the expression of “HA addition groups” to “HA-added group”.

5. Line 492: Delete “6. Patents” and the sentence below.

6. Further research has not been conducted to address the impact of humic acid on anaerobic digestion and biogas production, resulting in a lack of practical engineering applications. Also, some relevant references are suggested to discuss to state the novelty of this work, Journal of Hazardous Materials 2022, 438, 129556. Water Research 2023, 234, 119816.

7. Adjust the plot size so that the subplot in each main plot remain the same size.

The languages need to be improved.

Author Response

Prof. Marciana Barbulescu

Editor

Fermentation

Response: We sincerely appreciate your valuable feedback regarding our manuscript. In response to the reviewer's suggestions, we have revised the manuscript. All changes are highlighted in red font to facilitate the easy identification of these modifications

Responses to the reviewer’s comments:

Reviewer #1:

General Comments

Thank you for your positive comments on our manuscript. We agree with your suggestions and have revised the manuscript as described below in our detailed responses to your specific comments. All changes in the revised manuscript are highlighted in red font.

Specific Comments

Many thanks for your careful review and constructive suggestions regarding our manuscript. We are in complete agreement with your suggestions and have corrected all the errors you pointed out.

Line 125:Elaborate on the measurement steps of ROS in detail.

Thank you for your suggestion. The “the measurement steps of ROS in detail” has been added in Supplementary materials.

Line 256: Why does low concentration of humic acid promote the production of VFA? From Figure 2, it can be seen that low concentrations of humic acid still have a certain inhibitory effect on hydrolases.

Thank you for your question. (1) Although low concentrations of humic acid have a certain inhibitory effect on hydrolases, low concentrations of HA promote the activity of CoAT and BK during the AD process according to Fig. 2f and Fig. 2g. (2) Enzyme activity refers to the ability of enzyme molecules catalyzing chemical reactions, usually expressed in terms of the amount of substrate converted per unit time or Measured by the amount of product produced. When testing enzyme activity, certain conditions and systems are usually selected to simulate the activity of the enzyme in a specific environment. This can include factors such as temperature, pH, substrate concentration, etc. The detected enzyme activity only reflects the enzyme activity under specific conditions and cannot fully represent the activity level of the enzyme under other conditions. (3) The dominant bacterial phylum, including Firmicutes, Bacteroidetes, and Cloacimonetes, known for their ability to synthesize VFAs in AD processes, which contribute to the production of VFA.

Line 300: Change “which may be due to” to “possibly because of”.

Thank you for your suggestion. “Which may be due to” has been revised to “possibly because of” (Line 303).

Line 375: Change the expression of “HA addition groups” to “HA-added group”.

Thank you for your suggestion. The “HA addition groups” has been revised to “HA-added group” (Line 378).

Line 492: Delete “6. Patents” and the sentence below.

Thank you for your suggestion. The “6. Patents” and the sentence below have already been deleted.

Further research has not been conducted to address the impact of humic acid on anaerobic digestion and biogas production, resulting in a lack of practical engineering applications. Also, some relevant references are suggested to discuss to state the novelty of this work, Journal of Hazardous Materials 2022, 438, 129556. Water Research 2023, 234, 119816.

Thank you for your suggestion. All references suggested have been added.

Adjust the plot size so that the subplot in each main plot remain the same size.

Thank you for your suggestion. All plots have been adjusted.

Author Response File: Author Response.docx

Reviewer 2 Report

The manuscript entitled “Deeper insights into the effect of humic acid on kitchen waste anaerobic digestion: enzyme activities, microbial communitydynamics, and key metabolic pathways” by Lin Lyu and co-authors is focoused on the impact of humic acid on anaerobic digestion of kitchen waste. The data obtained are clearly described and I believe that the manuscript can be accepted for publication in Fermentation after minor revision. Please, find my comments below.

Please, provide the abbreviation list

The goal of the study has to be clearly emphasized in the introduction
Please, provide the data in the text in accordance with its accuracy (“The cumulative methane production for the CK, R1, R2, R3, R4, and R5 was 4537.99, 4239.16, 4269.25, 3856.31, 3745.37, and 3198.74 mL, respectively.” and etc)
I believe that researches provided by Efremenko et al. have to be cited.
The conclusions section should be written more extensively.

Author Response

Prof. Marciana Barbulescu

Editor

Fermentation

Responses to the reviewer’s comments:

Reviewer #2:

General Comments

The manuscript entitled “Deeper insights into the effect of humic acid on kitchen waste anaerobic digestion: enzyme activities, microbial community dynamics, and key metabolic pathways” by Lin Lyu and co-authors is focused on the impact of humic acid on anaerobic digestion of kitchen waste. The data obtained are clearly described and I believe that the manuscript can be accepted for publication in Fermentation after minor revision. Please, find my comments below.

Specific Comments

Many thanks for your careful review and constructive suggestions regarding our manuscript. We are in complete agreement with your suggestions and have corrected all the errors you pointed out.

The goal of the study has to be clearly emphasized in the introduction
Please, provide the data in the text in accordance with its accuracy (“The cumulative methane production for the CK, R1, R2, R3, R4, and R5 was 4537.99, 4239.16, 4269.25, 3856.31, 3745.37, and 3198.74 mL, respectively.” and etc)

Thank you for your suggestion. The data are provided below.

Time (day)	CK	R1	R2	R3	R4	R5
0	0.00	0.00	0.00	0.00	0.00	0.00
1	1314.66	1130.57	1098.31	1243.37	1006.82	959.93
2	2547.88	2353.93	2213.35	2393.18	1934.73	1764.48
3	3543.71	3252.79	3042.71	3154.51	2765.13	2376.27
4	4156.03	3909.00	3724.79	3652.78	3401.95	2967.89
5	4428.70	4160.48	4171.10	3809.28	3646.80	3182.03
7	4536.56	4237.40	4268.46	3855.04	3701.94	3197.90
9	4537.08	4237.71	4268.46	3855.30	3703.52	3198.46
11	4537.35	4237.71	4268.63	3855.47	3703.62	3198.51
13	4537.92	4238.27	4269.17	3856.26	3745.28	3198.74
15	4537.99	4239.16	4269.25	3856.31	3745.37	3198.74

I believe that researches provided by Efremenko et al. have to be cited.
The conclusions section should be written more extensively.

Thank you for your suggestion. All references suggested have been added.

Author Response File: Author Response.docx

Reviewer 3 Report

Scientific article entitled “Deeper insights into the effect of humic acid on kitchen waste anaerobic digestion: enzyme activities, microbial community dynamics, and key metabolic pathways” by authors Lin Lyu, Yanzeng Li, Shenghua Zhang and Zhou Chen is well written and interesting scientific article.

There are only minor issues that need clarification:

L87-88 please change „laboratory synthesized“ to „laboratory-prepared“ What raw material is used for production of kitchen waste? Did u compared composition of kitchen waste u prepared with average kitchen waste composition available locally?

L103 please add what alkali did u use to adjust pH to 7

L113 please give reference for the method used or describe the GC method used.

L119 please give conditions for the method used.

L126-128 please give description or reference for the determination of enzyme activities, since it is not clear what kits were used.

Author Response

Prof. Marciana Barbulescu

Editor

Fermentation

Responses to the reviewer’s comments:

Reviewer #3:

General Comments

Specific Comments

Many thanks for your careful review and constructive suggestions regarding our manuscript. We are in complete agreement with your suggestions and have corrected all the errors you pointed out.

L87-88 please change “laboratory synthesized “to “laboratory-prepared” What raw material is used for production of kitchen waste? Did u compared composition of kitchen waste u prepared with average kitchen waste composition available locally?

Thank you for your suggestion. The “laboratory synthesized” was revised to “laboratory-prepared” (Line 88). The main ingredients were rice (35%), meat (30%, including 12% of lean meat, 14% of soybeans and 4% of fish meat) and vegetables (35%, including equal amount of celery, spinach and cabbage). To ensure the homogeneity of the substrate, the kitchen waste used in this research was synthesized by selecting widely representative food ingredients based on the component analysis of our previous study “Chen, Z., Li, Y., Peng, Y., Ye, C., Zhang, S., 2021. Effects of antibiotics on hydrolase activity and structure of microbial community during aerobic co-composting of food waste with sewage sludge. Bioresour. Technol. 321, 124506”. References have also been added to the text. More details have been added (line 88-91).

L103 please add what alkali did u use to adjust pH to 7

Thank you for your suggestion. We use 5M NaOH to adjust pH to 7. This content has been added in line 110.

L113 please give reference for the method used or describe the GC method used.

Thank you for your suggestion. Reference has been added in line 121.

L119 please give conditions for the method used.

Thank you for your suggestion. More details are dilution added in line 122-123.

L126-128 please give description or reference for the determination of enzyme activities, since it is not clear what kits were used.

Thank you for your suggestion. More details about enzyme activities detection are added in line 137-140.

Author Response File: Author Response.docx

Reviewer 4 Report

Overview and general recommendation:

In this manuscript, the authors assessed the effect of humic acid (HA) levels on the anaerobic digestion (AD) process from kitchen waste. Methane and short-chain fatty acids (VFAs) production, enzyme activities, and microorganism and gene abundances, were analyzed under different HA concentrations. The paper's claimed importance is addressing a knowledge gap, providing a deeper analysis of the HA effect on the AD, and guiding the development and optimization of this process. Overall, this is a valuable contribution to the research area of anaerobic bioprocesses development. Furthermore, the manuscript is well-written and presented. Only a few major and minor aspects need to be addressed before the paper can be recommended for publication.

Major comments

i. Page 7, lines 241-250: The differences depicted in Figure 2 (e), (f), and (g) varying HA dosage do not seem to be statistically significant, at least considering the standard deviations (error bars). In other words, confidence intervals for the means are likely to overlap. Please, clarify this issue.

ii. Conclusions: The main research findings are summarized. However, according to the authors, what are the implications? Is increasing HA levels recommended or not for the AD process? I understand that it is not. Please, complement this section.

Minor comments

iii. Page 3, lines 108-109: I recommend replacing ‘continuous vibration’ with ‘continuous shaking’.

iv. Page 12, Figure 5: The last label ‘i’ in the title (line 408) should be “l”, right?

Author Response

Prof. Marciana Barbulescu

Editor

Fermentation

Responses to the reviewer’s comments:

Reviewer #4:

General Comments

Specific Comments

Many thanks for your careful review and constructive suggestions regarding our manuscript. We are in complete agreement with your suggestions and have corrected all the errors you pointed out.

Page 7, lines 241-250: The differences depicted in Figure 2 (e), (f), and (g) varying HA dosage do not seem to be statistically significant, at least considering the standard deviations (error bars). In other words, confidence intervals for the means are likely to overlap. Please, clarify this issue.

Thank you for your suggestion. According to individual effects analysis conducted by SPSS statistics 26, the activity of BK has significant, as the P Value of BK are all less than 0.05 among different groups in different days.

For AK and CoAT, their P value are more than 0.05, which means there is no significant among different groups in different days. However, in the article, we did not describe the significant difference in the data of AK, BK and CoAT, but described the changing trend of the data. We think this is still convincing.

AK
Time	Day1	Day5	Day9	Day15
P Value	0.933	0.968	0.947	0.082

BK
Time	Day1	Day5	Day9	Day15
P Value	0.000	0.000	0.000	0.000

CoAT
Time	Day1	Day5	Day9	Day15
P Value	0.393	0.118	0.036	0.018

Conclusions: The main research findings are summarized. However, according to the authors, what are the implications? Is increasing HA levels recommended or not for the AD process? I understand that it is not. Please, complement this section.

Thank you for your suggestion. Relevant content has been added in line 493-494.

Page 3, lines 108-109: I recommend replacing ‘continuous vibration’ with ‘continuous shaking’.

Thank you for your suggestion. The “continuous vibration” was revised to “continuous shaking” (Line 115).

Page 12, Figure 5: The last label ‘i’ in the title (line 408) should be “l”, right?

Thank you for your suggestion. Yes, that's a lowercase "L", and we have changed ‘i’ to ‘l’(Line 425).

Author Response File: Author Response.docx