Denitrification Capacity of Volatile Fatty Acids from Sludge Fermentation: Lab-Scale Testing and Full-Scale Assessment

Round 1

Reviewer 1 Report

The manuscript titled "Denitrification capacity of volatile fatty acids from sludge fermentation: lab-scale testing and full-scale assessment" provides a comprehensive investigation into the potential integration of recovered volatile fatty acids (VFAs) into biological nitrogen removal processes. The study explores the use of VFAs, the primary products of acidogenic fermentation of waste sludge, as a viable carbon source for denitrification in activated sludge processes. Through meticulous lab-scale testing and a full-scale assessment, the authors compare single VFAs, fermented liquid (FL) from a full-scale waste sludge fermenter, and commercial hydroalcoholic solutions.

The manuscript's abstract outlines the research methodology and key findings, emphasizing the denitrification rates of different VFAs and their potential to replace external carbon sources in wastewater treatment. Notably, acetic acid exhibits the highest denitrification rates, while a synthetic VFAs mixture and FL show enhanced denitrification rates compared to sole acetate. The manuscript also delves into the positive impact of FL dosage on nitrogen removal in activated sludge treatment, as evidenced by mass balances across a full-scale wastewater treatment plant.

The conclusion highlights the effectiveness of VFAs in promoting denitrification, particularly when using mixed VFAs substrates. The manuscript underscores the importance of evaluating VFAs dosage effects in full-scale wastewater treatment plants, a topic with limited coverage in existing literature. The efficiency of manometric batch-tests in monitoring heterotrophic biomass activity under anoxic conditions is also emphasized, providing valuable insights into the specific denitrification activity of activated sludge biomass within a short time span.

The manuscript concludes by suggesting avenues for further research, including the identification of intermediate compounds during the denitrification of C4-C5 volatile acids and exploring kinetic aspects as indirect carbon sources for denitrification. Overall, the manuscript appears to make a significant contribution to the understanding of utilizing VFAs for denitrification in wastewater treatment processes. 

1. Line 8, "Abstract A single paragrap"

Delete the "A single paragrap"

2. After reading the Introduction, I couldn't find any statement that highlights the novelty of your study. What is the novelty of your study?

3. The manuscript has been written using many old references (such as: van Rijn et al., 1996; Hallin et al., 1998; Hatziconstantinou et al., 1996; Pavan et al., 1998; Kujawa et al., 1999; Barber et al., 2000; Moser-Engeler et al., 1998), which indicates a lack of novelty in your study. Please replace them with more recent references.

4. Line 216; "3. Results"

Edit it to "3. Results and discussions"

5. Line 93; "...the municipal WWTP of Sesto San Giovanni (Milan area, Italy)."

Add geographical coordinates.

Author Response

Dear reviewer,

thanks for spending time in revising our manuscript. A response has been given in the attached file. I hope we correctly understood all the points, and that our responses were satisfying.

Reviewer 1

1. Line 8, "Abstract A single paragrap"Delete the "A single paragrap"

Deleted

2. After reading the Introduction, I couldn't find any statement that highlights the novelty of your study. What is the novelty of your study?

As novelty statement, the following sentence was added at the end of the introductory paragraph: “The novelty of this work relays in the proposal of a simple and effective procedure to monitor the evolution of the denitrification rate in full scale WWTPs as well as for the comparison of the denitrification potential of different carbon sources including locally produced fermented liquors”. Furthermore, lines 98-104 were added to address this issue: “The novelty of this work relays in the proposal of a simple and effective procedure to monitor the evolution of the denitrification rate in full scale WWTPs as well as for the comparison of the denitrification potential of different carbon sources including locally produced fermented liquids. Furthermore, more than 65% of the consulted literature regarding denitrification with VFAs was indeed published before 2010, indicating that more recent insights might be valuable for this topic”.

3. The manuscript has been written using many old references (such as: van Rijn et al., 1996; Hallin et al., 1998; Hatziconstantinou et al., 1996; Pavan et al., 1998; Kujawa et al., 1999; Barber et al., 2000; Moser-Engeler et al., 1998), which indicates a lack of novelty in your study. Please replace them with more recent references.

Old references were used as the basis for the VFAs utilization hierarchy. Nevertheless, taking into account your suggestions, more recent studies were integrated into the manuscript as well: Choi et al.(2021), Sapmaz et al.(2022), Li et al (2023).

4. Line 216; "3. Results" Edit it to "3. Results and discussions"

Changed.

5. Line 93; "...the municipal WWTP of Sesto San Giovanni (Milanarea, Italy)."Add geographical coordinates.

Added, together with the population equivalent.

Thanks again,

kind regards

the authors 

Reviewer 2 Report

Comments on fermentation-2783613-peer-review-v1：

The article presents a comprehensive study on the use of volatile fatty acids (VFAs) derived from waste sludge for enhancing the denitrification process in wastewater treatment. Mass balances across the full-scale wastewater treatment plant demonstrated the positive role of FL dosage in enhancing the denitrification process in the activated sludge treatment.My overall opinion is that this paper could be a interesting valuable paper if minor modifications are implemented.

1. For the selection of initial conditions, the reasons should be explained, and as far as possible close to the engineering parameters. Please clarify more clearly.

2. While the study references relevant literature, expanding the review to include a wider range of previous studies on similar applications of VFAs in denitrification could provide a more comprehen- sive context.

3. Some sections could benefit from more detailed explanations of methodologies, particularly regarding the selection and preparation of the VFAs and the criteria for assessing denitrification efficiency.

4. A more explicit comparison between traditional carbon sources and VFAs would enhance the understanding of VFAs' efficiency and cost-effectiveness.

5. Investigating the variability in VFA composition from different waste sludge sources and its impact on denitrification efficiency could be valuable.

6. An analysis of the environmental impacts, including potential greenhouse gas emissions during the VFA recovery process, would be beneficial.

7. The references should be expanded. Some new literatures might be help the authors to further deepen the understanding of reaction mechanism as well as newest developing in this field (Journal of Environmental Management, 2023, 326: 116790   Regeneration mechanism of a novel high-performance biochar mercury adsorbent directionally modified by multimetal multilayer loading).

Author Response

Dear reviewer,

thanks for spending time in revising our manuscript. A response has been given in the attached file. I hope we correctly understood all the points, and that our responses were satisfying.

Reviewer 2:

The article presents a comprehensive study on the use of volatile fatty acids (VFAs) derived from waste sludge for enhancing the denitrification process in wastewater treatment. Mass balances across the full-scale wastewater treatment plant demonstrated the positive role of FL dosage in enhancing the denitrification process in the activated sludge treatment.My overall opinion is that this paper could be a interesting valuable paper if minor modifications are implemented.

1. For the selection of initial conditions, the reasons should be explained, and as far as possible close to the engineering parameters. Please clarify more clearly.

At lines 181-183 of the revised manuscript an additional clarification was given explaining the reasoning behind the section of specific initial concentrations and COD/N ratios is expressed. Furthermore, reasons to explain why the trial is conducted under COD or N limitation are discussed. The following text was added: “These ranges were determined to be close to those of the WWTP operational conditions, and to obtain a significant number of experimental points for the subsequent elaborations.”

2. While the study references relevant literature, expanding the review to include a wider range of previous studies on similar applications of VFAs in denitrification could provide a more comprehen- sive context.

As requested, other studies were integrated into the manuscript: Choi et al.(2021), Sapmaz et al.(2022), Li et al (2023).

3. Some sections could benefit from more detailed explanations of methodologies, particularly regarding the selection and preparation of the VFAs and the criteria for assessing denitrification efficiency.

At lines 131-146 and154-156 of the revised manuscript more detailed methodology was added. Specifically, the following additional explanations were reported:

“The synthetic VFAs stock solutions were prepared at a concentration of 100 g COD L-1. Acetate and propionate solutions were prepared starting from the sodium salts, while for butyrate and valerate, butyric acid and valeric acid were used, and the ob-tained stock solution was stabilized with concentrated NaOH (3M). Sodium nitrate salt was used for the preparation of a stock solution at a concentration of 20 g N-NO3 L-1.”, “The molar cumulative amount was converted into molar nitrate denitrified over time and then into cumulative mass of nitrate denitrified.”.

4. A more explicit comparison between traditional carbon sources and VFAs would enhance the understanding of VFAs' efficiency and cost-effectiveness.

Indeed, we agreed on the relevancy of an economic assessment as a further step. Our experimentation has provided solid evidences on the fact that the fermentation liquid is fostering a faster denitrification process (see Fig. 5 and comment at lines 321-330), thus proving the feasibility of using this internally produced liquid as a C-source in substitution to conventional commercial solutions. Techno-economic analysis has been included as future development in the conclusion paragraph.

5. Investigating the variability in VFA composition from different waste sludge sources and its impact on denitrification efficiency could be valuable.

 The VFA composition of the FL of the studies cited in our work was added as comparison “In addition, wide ranges of single VFAs concentrations in the FL mixture used are reported by authors as, among the cited works, acetate varies in the range 15% to 35%, propionate in the range 15% to 30%, butyrate and iso-butyrate in the range 8% to  45%, valerate and iso-valerate in the range 5% to 20%, and caproate and iso-caproate in the range 0% to 45% “ (lines 66-70).

6. An analysis of the environmental impacts, including potential greenhouse gas emissions during the VFA recovery process, would be beneficial.

In lines 403-404, a reference to the expected reduction in GHG emissions was added: “Furthermore, the improved denitrification efficiency is usually related to the re-duction of greenhouse gas emissions as the production of N2O is reduced”. Furthermore, LCA analysis included as future development in the conclusion paragraph.

7. The references should be expanded. Some new literatures might be help the authors to further deepen the understanding of reaction mechanism as well as newest developing in this field (Journal of Environmental Management, 2023, 326: 116790 Regeneration mechanism of a novel high-performance biochar mercury adsorbent directionally modified by multimetal multilayer loading).

Thanks for suggesting this interesting paper. Nevertheless, we could not find a correlation with to the topic of the present study and therefore we did not include it in the references.

Thanks again,

kind regards

the authors 

Reviewer 3 Report

The work is well organized and presented. I suggest that the paper be published after a minor revision

Specific comments：

1. Line 8, “A single paragrap” should be deleted.

2. The second full spelling of SDNR appears on line 79.

3. The information in the introduction should be more specific.

4. The references are somewhat dated, it is recommended to consult more recent articles.

5. One or more references from Fermentation should be cited.

6. sCOD is not abbreviated in the article.

7. Line 274 should use the past passive voice.

8. There are some grammatical errors in the article, such as the tense of the passive voice.

9. The experimental scheme device diagram should be improved.

10. Please note the unit format of the text.

11. The data processing of the experiment part is lacking, so it is suggested to enhance the data processing to be more supportive.

Author Response

Dear reviewer,

thanks for spending time in revising our manuscript. A response has been given in the attached file. I hope we correctly understood all the points, and that our responses were satisfying.

Reviewer 3

1. Line 8, “A single paragrap” should be deleted.

Deleted

2. The second full spelling of SDNR appears on line 79.

Deleted

3. The information in the introduction should be more specific.

The introduction was revised in order to give a better and more complete overview of the cited literature and the link with the present study. For example, the following text was added: “Internal reuse of VFAs from sludge fermentation in the same WWTP is particularly interesting as VFAs recovery for external reuse might be challenging from the tech-nical and regulatory perspectives”, “. In addition, wide ranges of single VFAs concentrations in the FL mixture used are reported by authors as, among the cited works, acetate varies in the range 15% and to 35%, propionate in the range 15% and to 30%, butyrate and iso-butyrate in the range 8% -to  45%, valerate and iso-valerate in the range 5% -to 20%, and caproate and iso-caproate in the range 0% to- 45%”, “On the other hand, other authors [24,25] reported that propionate and butyrate might promote a faster denitrification in comparison to acetate due to a higher electron equivalent content.”, “The novelty of this work relays in the proposal of a simple and effective procedure to monitor the evolution of the denitrification rate in full scale WWTPs as well as for the comparison of the denitrification potential of different carbon sources including lo-cally produced fermented liquids. Furthermore, more than 65% of the consulted liter-ature regarding denitrification with VFAs was indeed published before 2010, indicat-ing that more recent insights might be valuable for this topic.” (58-60, 66-70, 84-86, 98-104)

4. The references are somewhat dated, it is recommended to consult more recent articles.

Old references were used as the basis for the VFAs utilization hierarchy. Nevertheless, taking into account your suggestions, more recent studies were integrated into the manuscript as well: Choi et al.(2021), Sapmaz et al.(2022), Li et al (2023).

5. One or more references from Fermentation should be cited.

  Nzeteu et al. ( 2022), and Giduthuri, et al. (2023) were added.

6. sCOD is not abbreviated in the article.

In line 246 of the revised manuscript the abbreviation is introduced.

7. Line 274 should use the past passive voice.

Changed in: “In addition, Figure 6 reports the VFAs percentage distribution of the FL used in trials T5-T9, as well as the average composition of FL samples collected at the WWTP.”

8. There are some grammatical errors in the article, such as the tense of the passive voice.

Some minor modifications in the English language were made.

9. The experimental scheme device diagram should be improved.

If the reference was to Fig. 3, some graphical modifications were performed. Furthermore, additional pieces of information were added to Fig.1.

10. Please note the unit format of the text.

The unit format was uniformed throughout the manuscript.

11. The data processing of the experiment part is lacking, so it is suggested to enhance the data processing to be more supportive.

At lines 131-146 and 154-156 of the revised manuscript more detailed methodology was added. Specifically, the following additional explanations were reported:

“The synthetic VFAs stock solutions were prepared at a concentration of 100 g COD L-1. Acetate and propionate solutions were prepared starting from the sodium salts, while for butyrate and valerate, butyric acid and valeric acid were used, and the ob-tained stock solution was stabilized with concentrated NaOH (3M). Sodium nitrate salt was used for the preparation of a stock solution at a concentration of 20 g N-NO3 L-1.”, “The molar cumulative amount was converted into molar nitrate denitrified over time and then into cumulative mass of nitrate denitrified.”.

Thanks again,

kind regards

the authors 

Round 2

Reviewer 1 Report

No further comments