A Comparative Study of Biological and Ozonation Approaches for Conventional and Per- and Polyfluoroalkyl Substances Contaminant Removal from Landfill Leachate

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

This manuscript addresses a timely and relevant topic: the removal of PFAS and conventional pollutants from landfill leachate using a combined biological and ozone-based treatment process. The experimental design is thorough, and the comparison between biological-only and hybrid processes is well executed. The findings, especially the unexpected release of PFAS following ozonation, offer valuable insight into potential drawbacks of ozone-based AOPs when dealing with PFAS-rich matrices. The article is suitable for publication after some clarifications and minor revisions.

1) The paper discusses reduced sludge production, but lacks any post-treatment characterization of biomass structure or microbial community changes after ozone exposure.

2) Standard deviations are provided for most parameters, but the manuscript lacks mention of how many replicates were conducted. Please specify the number of replicates for each measurement and whether statistical significance was tested between process periods.

3) The explanation of color removal efficiency after ozonation could benefit from a brief discussion of possible chromophoric structures present in landfill leachate (e.g., humic substances, aromatic compounds).

4) Consistently use "ozonation" instead of "ozone oxidation" for clarity.

5) Consider improving figure resolution for Figure 3 (PFAS removal efficiency).

6) Suggested Citation – Relevant Literature: I recommend that the authors cite the following article:https://doi.org/10.1016/j.jwpe.2020.101645. This suggested paper is relevant because it explores the removal of highly persistent halogenated organic contaminants from complex real wastewater matrices using reductive approaches. Although the target compounds (chlorinated benzenes) differ from PFAS, both studies tackle the challenge of degrading recalcitrant, toxic organohalogenated pollutants in real environmental waters. Including this citation would broaden the discussion of possible complementary or alternative treatment strategies for PFAS-contaminated leachates and emphasize the importance of process selection according to pollutant class and matrix composition.

In summary, the proposed manuscript presents important findings with clear environmental significance, especially concerning the risks and benefits of integrating ozonation into biological treatment processes for PFAS removal. With moderate revisions focused on clarity, mechanism discussion, and literature context, the article will provide a strong contribution to the field. Therefore, I recoomend the publication of proposed article after minor revisons.

Author Response

Reviewers' comments:

Reviewer #1:

This manuscript addresses a timely and relevant topic: the removal of PFAS and conventional pollutants from landfill leachate using a combined biological and ozone-based treatment process. The experimental design is thorough, and the comparison between biological-only and hybrid processes is well executed. The findings, especially the unexpected release of PFAS following ozonation, offer valuable insight into potential drawbacks of ozone-based AOPs when dealing with PFAS-rich matrices. The article is suitable for publication after some clarifications and minor revisions.

Reply – We would like to thank Reviewer 1 for her/his general positive comment on our study.

Specific comments:

1) The paper discusses reduced sludge production, but lacks any post-treatment characterization of biomass structure or microbial community changes after ozone exposure.

Reply – We agree with the Reviewer's suggestion that the post-treatment characterization of biomass structure or microbial community changes after ozone exposure would certainly be of interest. In our opinion, this characterisation is out of the scope of the present manuscript, and we prefer to avoid including it in the paper. We hope that the Reviewer can understand our point of view. The characterisation of the biomass growing in SBBGR and BIO-CHEM process, however, can be found in in our previous study (Chem. Eng. J. 2011, 168, 1085–1092, doi:10.1016/j.cej.2011.01.089.).

2) Standard deviations are provided for most parameters, but the manuscript lacks mention of how many replicates were conducted. Please specify the number of replicates for each measurement and whether statistical significance was tested between process periods.

Reply – We believe that the number of replicates and the statistical significance are meaningful, as they give the opportunity to the reader to see the statistical significance between process periods. Accordingly, in the revised version, we mentioned that the measurement of all parameters is done weekly throughout the entire operational period, providing average values and standard deviation for each period and each parameter (lines 216-207). We hope that our reply is welcomed by Reviewer 1.

3) The explanation of color removal efficiency after ozonation could benefit from a brief discussion of possible chromophoric structures present in landfill leachate (e.g., humic substances, aromatic compounds).

Reply –  We would like to thank the Reviewer for this comment. We have highlighted the proposed point (please check lines 440-447)

4) Consistently use "ozonation" instead of "ozone oxidation" for clarity.

Reply – We have changed "ozone oxidation" in the whole manuscript to “ozonation” as Reviewer 1 requested.

5) Consider improving figure resolution for Figure 3 (PFAS removal efficiency).

Reply – The resolution of Figure 3 was improved in the revised version of the manuscript, following the suggestion of Reviewer 1. We hope that its quality will be good enough.

6) Suggested Citation – Relevant Literature: I recommend that the authors cite the following article:https://doi.org/10.1016/j.jwpe.2020.101645. This suggested paper is relevant because it explores the removal of highly persistent halogenated organic contaminants from complex real wastewater matrices using reductive approaches. Although the target compounds (chlorinated benzenes) differ from PFAS, both studies tackle the challenge of degrading recalcitrant, toxic organohalogenated pollutants in real environmental waters. Including this citation would broaden the discussion of possible complementary or alternative treatment strategies for PFAS-contaminated leachates and emphasize the importance of process selection according to pollutant class and matrix composition.

Reply – Thank you for your valuable comment. We have taken her/his comment into consideration, and the suggested article has been cited in the revised manuscript (lines 101-105).

In summary, the proposed manuscript presents important findings with clear environmental significance, especially concerning the risks and benefits of integrating ozonation into biological treatment processes for PFAS removal. With moderate revisions focused on clarity, mechanism discussion, and literature context, the article will provide a strong contribution to the field. Therefore, I recommend the publication of the proposed article after minor revisions.

Reply – We would like to thank Reviewer 1 for indicating our paper to be accepted after minor revisions.

Author Response File: Author Response.pdf

Reviewer 2 Report

Comments and Suggestions for Authors

This manuscript investigated the degradation of medium-aged landfill leachate by using the Sequencing Batch Biofilter Granular Reactor (SBBGR) with and without ozone integration (BIO-CHEM process). Special attention is given to removing per- and polyfluoroalkyl substances (PFAS), a class of persistent and bioaccumulative pollutants. Although the theme of this manuscript is quite suitable for readers of Water, it lacks sufficient innovation and complete research content. The main problems are as follows:

1. The innovation of the BIO-CHEM process is insufficient, and there have been many related research reports (lines 88-104).
2. This manuscript lacks a detailed characterization of the key microbial communities that play a degrading role in the SBBGR system. Therefore, this manuscript can only briefly report the degradation of basic water quality parameters.
3. This manuscript did not conduct non-targeted and total fluorine determinations of PFAS, nor did it detect the degradation and transformation processes/products of PFAS in the SBBGR system and BIO-CHEM process.

A detailed review of the article's details is as follows:

1. Lines 52-59: This paragraph should provide an overview of the concentration levels of PFAS in landfill leachate (lines 52-54).

Next, the ecological or health hazards of PFAS in landfill leachate should be reviewed, rather than the hazards of PFAS in drinking water.

2. 2. Materials and methods: Section 2.1 should first introduce the chemicals and reagent materials used in this manuscript, or include them in the supplementary materials.
3. Fig. 1: The schematic diagram in Figure 1 is too simple, not clear and complete enough, and lacks many components. For example, the SBBGR system lacks a water distribution tank, an outlet tank, a pressure probe (line 155), etc. The ozone unit is lacking ozone generator, ozone destructor, UV ozone analyser, etc. In addition, sampling points need to be provided.
4. Section 2.2: How many sets of SBBGR and ozone oxidation units were run in this experiment, that is to say, whether all the operation data in this manuscript are parallel or repetitive.
5. Lines 170-171: Detailed water quality parameters of the landfill leachate need to be provided.
6. Lines 179-185: Why are only these 13 types of PFAS measured? What is the basis for the selection?
7. Lines 199-213: The injection volume of HPLC is 100 microliters (line 199), which is quite large. Has sample pretreatment been carried out on the influent and effluent of the landfill leachate, such as filtration, concentration, SPE, etc.?

Please also provide 13 PFAS mass spectrometry detection parameters, such as parent ions, daughter ions, collision energy, retention time, etc. as well as quality assurance and quality control parameters, such as recovery rate, detection limit, etc.

8. Lines 256-259: The trend concentrations of TSS and VSS showed regressive behaviour in the influent sample. Please provide some reasonable explanations.
9. Line 265: Table 1 summarizes the average concentrations of conventional pollutants. What is the average of the data here? Is it the average of several days, tens of days or several months of data? Please also provide an explanation in Table 1. n=?
10. Lines 318-321: Whether the residual ozone needs to be decomposed, whether it enters the SBBGR system, and whether it has an impact on microorganisms?
11. Line 411: Table 2 reports the average concentrations of PFAS compounds. The average problem here is the same as that of question 9.
12. Lines 451-455: For a high release of most PFAS compounds (Figure 3), Is there another possibility that the ozone oxidation process has degraded the PFAS precursors, leading to an increase in the concentrations of the 13 detected PFAS? Therefore, it is necessary to conduct non-targeted PFAS screening and total fluorine determination on the samples before and after the treatment of landfill leachate by SBBGR system and BIO-CHEM process.
13. Lines 456-460: Whether residual ozone is toxic to microorganisms and leads to a decline in their degradation performance, the issue here is the same as that in question 10.
14. This manuscript is highly redundant due to the lack of key experimental content such as microbial characterization and PFAS identification, with a considerable amount of space devoted to describing simple water quality parameters.

Comments on the Quality of English Language

The English could be improved to more clearly express the research.

Author Response

Reviewers' comments:

Reviewer #2:

This manuscript investigated the degradation of medium-aged landfill leachate by using the Sequencing Batch Biofilter Granular Reactor (SBBGR) with and without ozone integration (BIO-CHEM process). Special attention is given to removing per- and polyfluoroalkyl substances (PFAS), a class of persistent and bioaccumulative pollutants. Although the theme of this manuscript is quite suitable for readers of Water, it lacks sufficient innovation and complete research content. The main problems are as follows:

1. The innovation of the BIO-CHEM process is insufficient, and there have been many related research reports (lines 88-104).

Reply – We appreciate the reviewer’s comment and the opportunity to clarify the novelty of our work. While it is true that several studies have addressed aspects of the BIO-CHEM process, our research introduces significant advancements that distinguish it from prior work. Specifically, the proposed technology has not been previously applied to the treatment of PFAS pollutants. Additionally, only a limited number of studies have investigated the use of the BIO-CHEM process for leachate treatment. However, those few studies did not provide an in-depth evaluation of the process’s effectiveness in removing a broad spectrum of pollutants, including emergent contaminants. Our study addresses this gap by comprehensively assessing the performance of the BIO-CHEM process in treating complex contaminant mixtures, thereby contributing novel insights into its potential and practical applicability.

2. This manuscript lacks a detailed characterization of the key microbial communities that play a degrading role in the SBBGR system. Therefore, this manuscript can only briefly report the degradation of basic water quality parameters.

Reply – We thank the Reviewer for the valuable suggestion concerning the detailed characterization of the key microbial communities. While we acknowledge its potential interest, we consider this aspect beyond the scope of the current study and have therefore opted not to include it. We kindly ask for the Reviewer's understanding in this regard.

3. This manuscript did not conduct non-targeted and total fluorine determinations of PFAS, nor did it detect the degradation and transformation processes/products of PFAS in the SBBGR system and BIO-CHEM process.

Reply – We thank the Reviewer for highlighting the importance of non-targeted and total fluorine analyses, as well as the identification of PFAS degradation and transformation products. We fully agree that these aspects would provide a more comprehensive understanding of PFAS behavior in the SBBGR system and BIO-CHEM process. However, these analyses were beyond the scope and objectives of the current study, which focused primarily on the removal efficiency of the most produced and monitore PFAS in the world. We consider these suggested investigations highly valuable and will certainly take them into account for future research efforts.

A detailed review of the article's details is as follows:

1. Lines 52-59: This paragraph should provide an overview of the concentration levels of PFAS in landfill leachate (lines 52-54). Next, the ecological or health hazards of PFAS in landfill leachate should be reviewed, rather than the hazards of PFAS in drinking water.

Reply – In response to the Reviewer’s comment, we have included an extensive and comprehensive overview of PFAS concentration levels in landfill leachate and their associated ecological and human health risks (lines 54-75 in the revised version).

2. Materials and methods: Section 2.1 should first introduce the chemicals and reagent materials used in this manuscript, or include them in the supplementary materials.

Reply – The Reviewer is completely right, and we apologize for the missing information regarding the chemicals and reagent materials used. We have included them in the supplementary materials, and we have mentioned this indication in lines 223-224 of the revised version.

3. 1: The schematic diagram in Figure 1 is too simple, not clear, and complete enough, and lacks many components. For example, the SBBGR system lacks a water distribution tank, an outlet tank, a pressure probe (line 155), etc. The ozone unit is lacking ozone generator, ozone destructor, UV ozone analyser, etc. In addition, sampling points need to be provided.

Reply – Figure 1 has been reorganized as requested by Reviewer 2.

4. Section 2.2: How many sets of SBBGR and ozone oxidation units were run in this experiment, that is to say, whether all the operation data in this manuscript are parallel or repetitive.

Reply – We thank the reviewer for this important question. In this study, one set of the SBBGR (Sequencing Batch Biofilm Granular Reactor) and one ozone oxidation unit were operated continuously during the entire experimental period. Influent and effluent samples were collected weekly and analyzed promptly after each sampling to ensure data reliability and consistency. The operation data presented in the manuscript therefore represent a single system operated over time, and not parallel or replicate systems. While the data are not derived from parallel reactors, the weekly sampling and repeated measurements over an extended period (i.e., multiple operational cycles) provide temporal replication. This approach allowed us to observe consistent trends and evaluate system performance.

5. Lines 170-171: Detailed water quality parameters of the landfill leachate need to be provided.

Reply – The authors are not sure they properly understand this comment from the reviewer. SBBGR and BIO-CHEM process performances were characterized in terms of the most widely used quality parameters, including also the emerging contaminants such as PFAS compounds

 

6. Lines 179-185: Why are only these 13 types of PFAS measured? What is the basis for the selection?

Reply – Thank you for your insightful question. The selection of the 13 PFAS compounds analyzed in this study was based on a combination of regulatory relevance, environmental prevalence, and analytical feasibility. Specifically, these compounds are among the most commonly monitored and reported PFAS species in international environmental monitoring programs, including those prioritized by the U.S. EPA (e.g., Method 537.1), the EU Water Framework Directive, and OECD guidelines. Moreover, these 13 PFAS represent a mix of legacy and emerging compounds, including both perfluoroalkyl carboxylic acids and perfluoroalkane sulfonic acids, allowing for a representative assessment of PFAS contamination patterns. They were also selected based on the availability of high-quality analytical standards and validated quantification methods to ensure robust and reproducible results. While we acknowledge that there are hundreds of known PFAS compounds, our focus on these 13 provides meaningful insight into environmental risks while maintaining analytical reliability.

7. Lines 199-213: The injection volume of HPLC is 100 microliters (line 199), which is quite large. Has sample pretreatment been carried out on the influent and effluent of the landfill leachate, such as filtration, concentration, SPE, etc.?

Please also provide 13 PFAS mass spectrometry detection parameters, such as parent ions, daughter ions, collision energy, retention time, etc. as well as quality assurance and quality control parameters, such as recovery rate, detection limit, etc.

Reply – Thank you for the comment. The text has been revised to clarify the sample preparation and data analysis procedures (lines 228-258). Specifically, it now includes details on the centrifugation step (5000 rpm, 5 min), the rationale behind performing three levels of dilution (1:10, 1:100, 1:1000) to ensure all PFAS compounds fall within the calibration range, and the use of EICC (m/z width 0.02) for quantification. It also explains that recoveries were not evaluated due to the absence of an extraction step, as large-volume injection was employed. Additionally, Table 1 has been updated with a new column reporting the LOQ values for each PFAS.

8. Lines 256-259: The trend concentrations of TSS and VSS showed regressive behaviour in the influent sample. Please provide some reasonable explanations.

Reply – We appreciate the reviewer’s observation. As noted, the trend of TSS and VSS in the influent showed a regressive (declining) behavior over time. This trend can be reasonably attributed to the fact that the leachate used in our study was collected from a relatively stabilized landfill site. Over time, as the landfill matures, the organic load and suspended solids typically decrease due to ongoing natural biodegradation and a reduction in the input of fresh waste. This can lead to lower TSS and VSS levels in the influent.

9. Line 265: Table 1 summarizes the average concentrations of conventional pollutants. What is the average of the data here? Is it the average of several days, tens of days or several months of data? Please also provide an explanation in Table 1. n=?

Reply – We agree with the Reviewer's suggestion. In the revised version, we mentioned that the measurement of all parameters is done weekly throughout the entire operational period, providing average values and standard deviation for each period and each parameter (lines 216-217).

10. Lines 318-321: Whether the residual ozone needs to be decomposed, whether it enters the SBBGR system, and whether it has an impact on microorganisms?

Reply – We thank the reviewer for this important and constructive comment. The ozonation step is performed in a separate compartment downstream of the SBBGR. To prevent ozone from entering the biological system, the ozonated effluent is fully degassed and allowed sufficient retention time to ensure the decomposition of residual ozone before any recirculation. This precaution effectively minimizes the risk of ozone carryover. Furthermore, since the ozone-treated effluent is not fed back into the SBBGR without prior decomposition of residual ozone, no adverse effects on microbial activity were observed. In fact, the system maintained stable COD and NH₃ removal efficiencies throughout the operation, indicating healthy microbial performance.

11. Line 411: Table 2 reports the average concentrations of PFAS compounds. The average problem here is the same as that of question 9.

Reply – In the revised version, we mentioned that the measurement of all parameters (PFAS and conventional pollutants) is done weekly throughout the entire operational period, providing average values and standard deviation for each period and each parameter (lines 216-217).

12. Lines 451-455: For a high release of most PFAS compounds (Figure 3), Is there another possibility that the ozone oxidation process has degraded the PFAS precursors, leading to an increase in the concentrations of the 13 detected PFAS? Therefore, it is necessary to conduct non-targeted PFAS screening and total fluorine determination on the samples before and after the treatment of landfill leachate by SBBGR system and BIO-CHEM process.

Reply – We would like to thank the reviewer for highlighting this important issue concerning the potential transformation of PFAS precursors during treatment and its impact on PFAS monitoring. We acknowledge that ozone oxidation, particularly in the BIO-CHEM process integrated with the SBBGR, can degrade PFAS precursors. This leads to the formation of certain terminal PFAS compounds and an apparent increase in their concentrations, as illustrated in Figure 3. This is a well-recognised and plausible phenomenon, as ozone and other advanced oxidation processes can transform non-targeted precursor compounds into shorter-chain or terminal perfluoroalkyl acids (PFAAs), which are more stable and commonly detected in targeted analyses. However, in our study, PFAS compunds were measured at inlet and outlet of ozone reactor and no significant variations in concentrations were observed. Therefore, the increase of PFAS concentration in the effluent should be ascribed to their release from SBBGR biomass.

13. Lines 456-460: Whether residual ozone is toxic to microorganisms and leads to a decline in their degradation performance, the issue here is the same as that in question 10.

Reply – We appreciate the reviewer’s follow-up comment regarding the potential toxicity of residual ozone to microorganisms and its impact on degradation performance. As noted in our response to comment number 10, in our system configuration, ozone is applied downstream of the SBBGR, and no direct contact between residual ozone and the biological reactor occurs. However, we agree that the toxicity of residual ozone is an important consideration when designing integrated treatment systems.

14. This manuscript is highly redundant due to the lack of key experimental content such as microbial characterization and PFAS identification, with a considerable amount of space devoted to describing simple water quality parameters.

Reply – We thank the reviewer for the continued evaluation and thoughtful feedback. As noted in our earlier response, our study aims to contribute novel insights into the application of the BIO-CHEM process, particularly for PFAS-containing leachate treatment, an area that remains largely unexplored in current literature. While we acknowledge that several water quality parameters are presented in detail, this was done deliberately to establish a comprehensive understanding of baseline treatment performance across conventional and emerging contaminants under real leachate conditions. We hope this response can be welcomed by the reviewer.

Author Response File: Author Response.pdf

Reviewer 3 Report

Comments and Suggestions for Authors

This manuscript presents a comparative study on the performance of the Sequencing Batch Biofilter Granular Reactor (SBBGR) system with and without ozone integration (the BIO-CHEM process) for the removal of conventional pollutants (COD, TSS, VSS, color) and PFAS from middle-aged landfill leachate. Since landfill leachate is considered as a one of significant sources of PFAS contamination, developing effective remediation processes is indeed a relevant and timely research topic with practical implications.

However, I find that the majority of the results and discussion sections focus on the removal of conventional pollutants, which, as outlined in the introduction, have already been well studied and do not present much novelty. In contrast, the treatment of PFAS, the most critical and unique aspect of this study, is addressed only superficially. The discussion is limited to average values over the study period, without sufficient insight into the temporal or operational variability of PFAS removal.

Currently, the manuscript does not provide novel or useful findings regarding PFAS remediation. Nonetheless, if the authors could reconstruct the manuscript to center around a more detailed analysis of PFAS removal (for example, by investigating seasonal variations in PFAS concentrations and removal efficiencies, and correlating those with operational factors) the study could offer meaningful insights for the journal’s readers.

Author Response

Reviewers' comments:

Reviewer #3:

This manuscript presents a comparative study on the performance of the Sequencing Batch Biofilter Granular Reactor (SBBGR) system with and without ozone integration (the BIO-CHEM process) for the removal of conventional pollutants (COD, TSS, VSS, color) and PFAS from middle-aged landfill leachate. Since landfill leachate is considered as a one of significant sources of PFAS contamination, developing effective remediation processes is indeed a relevant and timely research topic with practical implications.

However, I find that the majority of the results and discussion sections focus on the removal of conventional pollutants, which, as outlined in the introduction, have already been well studied and do not present much novelty. In contrast, the treatment of PFAS, the most critical and unique aspect of this study, is addressed only superficially. The discussion is limited to average values over the study period, without sufficient insight into the temporal or operational variability of PFAS removal.

Currently, the manuscript does not provide novel or useful findings regarding PFAS remediation. Nonetheless, if the authors could reconstruct the manuscript to center around a more detailed analysis of PFAS removal (for example, by investigating seasonal variations in PFAS concentrations and removal efficiencies, and correlating those with operational factors) the study could offer meaningful insights for the journal’s readers.

 

Reply – The authors do not agree with the reviewer’s comment. SBBGR, an advanced biological system characterized by biomass enriched with slow-growing microorganisms, has been shown to achieve complete elimination of long-chain PFAS compounds (i.e., PFOA, PFHpA, PFOS, 6:2 FTSA, and PFHxS).

To the authors’ knowledge, this is the first experimental evidence of PFAS removal by a biological process.

Author Response File: Author Response.pdf

Round 2

Reviewer 2 Report

Comments and Suggestions for Authors

1. The authors only addressed some of the reviewers' questions. They did not conduct additional experiments for the characterization of the main microbial community in SBBGR and the non-targeted screening analysis of PFAS. Please include these two suggestions in the section 4 Conclusion and Future Directions and provide a future outlook.
2. line 63: When PFBS and PFBA first appear, their full names should be given.
3. lines 317-327: The description of the leachate treatment process for period C (3 months) does not correspond well to the schematic diagram in Figure 1. The BIO-CHEM process should involve first SBBGR treatment and then ozonation treatment. However, the text states that it is chemical + biological oxidation. Therefore, we are somewhat confused by the textual description and the leachate treatment process shown in Figure 1.
4. lines 339-341: This paragraph mentions 4 perfluorosulfonic acids, but the parentheses contain 5 compounds.
5. lines 549-559: Please also provide 13 PFAS mass spectrometry detection parameters, such as parent ions, daughter ions, collision energy, retention time, etc. as well as quality assurance and quality control parameters, such as recovery rate, detection limit, etc.
6. table 1: Table 1 only provides the removal rate of chroma, but no original data of the Influent and Effluent is given. That is, what are the absorbance values at 426, 556 and 660 nm, respectively?
7. line 919: The results showed that, among PFAS compounds, PFBS, PFOA and PFPA were the predominant compounds. PFPA should be PFBA. Please carefully review the main text content.

Comments on the Quality of English Language

The English could be improved to more clearly express the research.

Author Response

Please see the attachment

Author Response File: Author Response.docx