Iron Oxide-Bearing Wastes as Media for Supporting Biodegradation of BTEX

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

The paper investigates the use of iron oxide-bearing wastes, specifically drinking water treatment residuals (WTS) and mine water treatment sludge (MWTS), as media for the bioremediation of BTEX (benzene, toluene, ethylbenzene, and xylene) contaminated wastewater. The study focuses on determining the effectiveness of these wastes, with and without modification, in supporting the biodegradation of BTEX under both aerobic and anaerobic conditions. This study offers a new perspective on the treatment of BTEX. However, some issues exist in the manuscript. Therefore, the reviewer believes the manuscript could be considered for publication in "Minerals," after some revisions. Some specific comments are below:

(1)   Line 37: The use of first-person perspective needs to be revised, and the entire text should be checked for consistency.

(2)   Lines 48-50: Lack of references.

(3)   Lines 61-67: The novelty of this study is not sufficiently highlighted.

(4)   Line 76: Where did the wastewater treatment sludge come from, and when was the sampling conducted?

(5)   Were the experiments in the paper conducted only once or three times? Why are there no error bars?

(6)   Part 3.1: The discussion should be more detailed, particularly regarding the adsorption mechanism, especially the mass transfer process.

(7)   Similarly, Parts 3.2 and 3.3: A more detailed discussion of the mechanisms is required.

(8)   Other parts are well-written. The paper needs an additional section before the conclusion that discusses the global implications (environmental, current research impact, economic factors, applicability, etc.).

Comments on the Quality of English Language

Minor editing of English language required.

Author Response

The paper investigates the use of iron oxide-bearing wastes, specifically drinking water treatment residuals (WTS) and mine water treatment sludge (MWTS), as media for the bioremediation of BTEX (benzene, toluene, ethylbenzene, and xylene) contaminated wastewater. The study focuses on determining the effectiveness of these wastes, with and without modification, in supporting the biodegradation of BTEX under both aerobic and anaerobic conditions. This study offers a new perspective on the treatment of BTEX. However, some issues exist in the manuscript. Therefore, the reviewer believes the manuscript could be considered for publication in "Minerals," after some revisions. Some specific comments are below:

Comment 1:  Line 37: The use of first-person perspective needs to be revised, and the entire text should be checked for consistency.

Response 1: The authors have revised the manuscript suitably to address this comment.

Comment 2:   Lines 48-50: Lack of references.

Response 2: The authors have included two references (Ambaye et al., Chemosphere, 2022 and Elgazali et al., Microorganisms, 2023).

Comment 3:   Lines 61-67: The novelty of this study is not sufficiently highlighted.

Response 3: The authors have added appropriate text in the revised manuscript from lines 67-71.

Comment 4:   Line 76: Where did the wastewater treatment sludge come from, and when was the sampling conducted?

Response 4: As mentioned in the submitted manuscript the wastewater treatment sludge was collected from the Cardiff wastewater treatment works. A few more details have been included (revised manuscript line 85).

Comment 5:   Were the experiments in the paper conducted only once or three times? Why are there no error bars?

Response 5: As mentioned in the methods sections, all the studies were carried out in duplicates. Thus, the S.D error bars would not be meaningful and have therefore not shown in the graphs.

Comment 6:  Part 3.1: The discussion should be more detailed, particularly regarding the adsorption mechanism, especially the mass transfer process.

Response 6: The authors have included a brief discussion on adsorption mechanisms as suggested by the reviewer. (revised manuscript line 179-201).

Comment 7:   Similarly, Parts 3.2 and 3.3: A more detailed discussion of the mechanisms is required.

Response 7: The authors have included a brief discussion as suggested by the reviewer.

Comment 8:   Other parts are well-written. The paper needs an additional section before the conclusion that discusses the global implications (environmental, current research impact, economic factors, applicability, etc.).

Response 8: The authors have included a section on the significance of the study (revised manuscript line 480-486)

Reviewer 2 Report

Comments and Suggestions for Authors

The presence of BTEX compounds (benzene, toluene, ethylbenzene, and xylene) in groundwater is a pressing environmental concern due to their toxicity and persistence. To address this contamination, various treatment methods have been developed, including traditional pump-and-treat systems, enhanced bioremediation techniques, and more recent strategies like Permeable Reactive Barriers (PRBs) and In Situ Chemical Oxidation (ISCO).

 

There’s growing interest in innovative approaches, such as using iron oxide-bearing wastes, which show promise as cost-effective and sustainable solutions for BTEX remediation. In this context, the article investigates the efficacy of employing specific iron oxide-bearing wastes—namely drinking water treatment residuals and passive mine water treatment sludge—for biodegrading BTEX compounds. The aim of this study is to explore whether these waste materials can be viable, economical options for environmental remediation. However, I do think there are a few areas in which the manuscript could be strengthened before it’s ready for publication.

 

Firstly, while the manuscript mentions the physicochemical properties of the sludges used (MWTS, WTS, MX), I feel that providing a more detailed breakdown of these properties (like surface area, porosity, and mineral composition) would be beneficial. Adding further characterization techniques, such as BET surface area analysis and X-ray diffraction (XRD), would offer a clearer understanding of how these structural properties relate to BTEX sorption and degradation. It would also be helpful if the authors could clarify their rationale for selecting these specific methods.

 

Additionally, the study does mention the sorption of BTEX by the sludges, but I believe the manuscript could benefit from a clearer explanation of the experimental conditions used, such as temperature, pH, and mixing rates during sorption tests. Providing these details would ensure reproducibility and help the reader understand how these factors influence the sorption efficiency. It’s also important to explain why these particular experimental parameters were chosen and how they are expected to impact the outcomes.

 

Regarding the use of GC-MS for BTEX quantification, the manuscript could be improved by including more information on how the calibration curve was developed and validated. Specifically, details on the calibration range, limits of detection, and limits of quantification would enhance the reliability of the results. Justifying the chosen calibration range and its relevance to the experimental conditions would also strengthen this section.

 

The discussion on changes in microbial communities pre- and post-BTEX degradation is interesting; however, it seems to lack quantitative analysis of microbial growth (e.g., microbial counts or biomass measurements). Supplementing the data with quantitative PCR or microbial plating results could offer better insights into how microbial populations change over the course of BTEX degradation. Additionally, it would be great if the authors could explain their selection of microbial analysis methods to provide further clarity.

 

Lastly, although the study briefly mentions that the sludges effectively degrade BTEX over time, I suggest conducting additional tests on the long-term stability and reusability of these sludges over multiple cycles. This would provide more practical insights into their real-world applications. The authors could also elaborate on how they determined the number of cycles and their rationale for the degradation test time frame.

 

 

Author Response

Comment 1: Firstly, while the manuscript mentions the physicochemical properties of the sludges used (MWTS, WTS, MX), I feel that providing a more detailed breakdown of these properties (like surface area, porosity, and mineral composition) would be beneficial. Adding further characterization techniques, such as BET surface area analysis and X-ray diffraction (XRD), would offer a clearer understanding of how these structural properties relate to BTEX sorption and degradation. It would also be helpful if the authors could clarify their rationale for selecting these specific methods.

Response 1: The authors thank the reviewer for the suggestion. The details about the mineral compositions, XRD, porosity etc. have been reported in a previous study -Srivastava et al., Journal of Environmental Management, 2022. The rationale for specific methods was based on experience from our previous published works on iron sludges.

Comment 2: Additionally, the study does mention the sorption of BTEX by the sludges, but I believe the manuscript could benefit from a clearer explanation of the experimental conditions used, such as temperature, pH, and mixing rates during sorption tests. Providing these details would ensure reproducibility and help the reader understand how these factors influence the sorption efficiency. It’s also important to explain why these particular experimental parameters were chosen and how they are expected to impact the outcomes.

Response 2: The authors thank the reviewer for their comment. The vials with 0.25 g dried sludge in 21 ml BTEX wastewater were shaken or mixed at 150 rpm (line 105).

Comment 3: Regarding the use of GC-MS for BTEX quantification, the manuscript could be improved by including more information on how the calibration curve was developed and validated. Specifically, details on the calibration range, limits of detection, and limits of quantification would enhance the reliability of the results. Justifying the chosen calibration range and its relevance to the experimental conditions would also strengthen this section.

Response 3: The authors thank the reviewer for their comment. The authors have added the details from line 143-145 of the revised manuscript.

Comment 4: The discussion on changes in microbial communities pre- and post-BTEX degradation is interesting; however, it seems to lack quantitative analysis of microbial growth (e.g., microbial counts or biomass measurements). Supplementing the data with quantitative PCR or microbial plating results could offer better insights into how microbial populations change over the course of BTEX degradation. Additionally, it would be great if the authors could explain their selection of microbial analysis methods to provide further clarity.

Response 4: The authors would like to think the reviewer for their constructive feedback and for highlighting areas that could enhance the understanding of microbial dynamics in our study. The authors agree that a quantitative analysis of microbial growth could provide additional insights. However, Quantitative PCR (qPCR) and microbial plating are beyond the intended scope of this study. The authors primary focus was on profiling microbial community structure and composition changes, rather than on quantifying biomass or growth metrics. The authors selected high-throughput sequencing for its ability to capture broad microbial diversity and community shifts in response to BTEX degradation. This approach aligns with our goal to explore community-level responses rather than to measure absolute microbial counts. While not included in this study, the authors agree that qPCR and microbial plating could be valuable for future work aimed at quantifying microbial growth in similar systems.

Comment 5: Lastly, although the study briefly mentions that the sludges effectively degrade BTEX over time, I suggest conducting additional tests on the long-term stability and reusability of these sludges over multiple cycles. This would provide more practical insights into their real-world applications. The authors could also elaborate on how they determined the number of cycles and their rationale for the degradation test time frame.

Response 5: The authors thank the reviewer for their suggestions, however as this is a preliminary study the aim was to only highlight the potential. The authors plan on carrying out detailed work in the future along the lines that the reviewer has suggested.

Reviewer 3 Report

Comments and Suggestions for Authors

The ms by Al-Obaidi et al. explores the feasibility and interest of using Iron oxide-bearing sludges as media (source of bacterial degraders and nutrients, including Fe(III)) for the biodegradation of BTEX.  The manuscript is well written. The analysis of the changes in microbial communities before and after BTEX addition (over 160 days of incubation) constitutes a large part of the study. The authors lengthy describe the difference at the Phyla level, so at the highest level, and do not develop sufficiently on the iron-reducing aspect, which is at the centre of this study given the nature of the sludges.

Personal comment: the N aspect has not been discussed and I wonder if it is possible that the Mine Water Treatment Sludge and the mix (MX) might contain an important amount of N (from blasting with ammonium nitrate) that could explain in part the better performance of biotransformation compared to WTS?

Comments:

Section 2.1, lines 76-77: This sentence “WTS and MWTS were mixed with autoclaved wastewater treatment sludge (WWS) in a mass ratio of 9:1 (MX) were used for this study’’. Isn’t it ‘’ WTS, MWTS and a mix of MWTS with autoclaved wastewater treatment sludge (WWS) at a mass ratio of 9:1 (MX) …’’?

Lines 167-168: stated ‘’ Fe was the most abundant element in MWTS (36.5% DW), followed by Ca (1.8% DW), while WTWS had lower Fe and Ca’’. Please provide the ‘lower’ numbers, particularly for Fe, since there is no Table for metal content.

Lines 166-168: ‘’WTWS’. Elsewhere abbreviated as ‘WTS’.

Lines 172-173: “The amount of adsorbed contaminant per gram of absorbent sludge with respect to contact time is shown in Figure 1”. It seems that Figure 1 shows the concentration of BTEX in mg/L (starting initially at 45 g/L each) over 30 days.

Lines 174-175: ‘’The concentration of BTEX in the control remained almost constant’’. What is the control made of?

Lines 225-226: It is stated ‘NWTS exhibited a distinct and a higher lag period before the onset of BTEX degradation and was also the least effective media for degradation of BTEX’’. It does not seem to align with degradation rates presented in Table 1.

Line 273: the temperature in the lab varied from 1 °C to 24.5 °C? How come?

Lines 277-278: It is stated ‘’No correlation was observed between temperature and BTEX degradation’’. It seems unlikely. How was it interpreted?

Section 3.4. Microbial community changes: Too vague. Too long. Not informative enough. What is of special interest are the genera associated with Fe cycling, with aromatic compounds, and some others, but not all. Bacteroides, Firmicutes, and Proteobacteria cover so many different types of microorganisms.

Keep more emphasis on the genera Gallionella, Rhodoferax, Geobacter, and Geothrix, and aromatic degraders. Is there any relationship in what you observed in the distribution of iron-reducers and iron-oxidizers and the results of the Fe analysis?

Figure 5B. Some OTUs could be regrouped together as ‘others’.

Line 391: it is mentioned ‘WWS was added to enhance the nutritive value of MWTS’ Please include that sentence earlier in the text to explain why you did that mix.

Author Response

Comment 1: The ms by Al-Obaidi et al. explores the feasibility and interest of using Iron oxide-bearing sludges as media (source of bacterial degraders and nutrients, including Fe(III)) for the biodegradation of BTEX.  The manuscript is well written. The analysis of the changes in microbial communities before and after BTEX addition (over 160 days of incubation) constitutes a large part of the study. The authors lengthy describe the difference at the Phyla level, so at the highest level, and do not develop sufficiently on the iron-reducing aspect, which is at the centre of this study given the nature of the sludges.

Response 2: The authors agree with the reviewer’s contention that the iron-reducing microbial consortium are likely key organisms in the biodegradation observed, but because they don’t have any direct evidence of the activity of the organisms, they cannot at this stage of the research elucidate the extent of their role in the biodegradation and therefore thought it best to not speculate. They hope that future studies will address this key point.

Comment 3: Personal comment: the N aspect has not been discussed and I wonder if it is possible that the Mine Water Treatment Sludge and the mix (MX) might contain an important amount of N (from blasting with ammonium nitrate) that could explain in part the better performance of biotransformation compared to WTS?

Response 3: Agreed – this is an interesting aspect for further study – the authors thank the reviewer for this observation.

Comments:

Comments 4: Section 2.1, lines 76-77: This sentence “WTS and MWTS were mixed with autoclaved wastewater treatment sludge (WWS) in a mass ratio of 9:1 (MX) were used for this study’’. Isn’t it ‘’ WTS, MWTS and a mix of MWTS with autoclaved wastewater treatment sludge (WWS) at a mass ratio of 9:1 (MX) …’’?

Response 4: Thank you for highlighting this error. The authors have made the changes and is highlighted in red.

Comment 5: Lines 167-168: stated ‘’ Fe was the most abundant element in MWTS (41.4% DW), followed by Ca (2.37% DW), while WTWS had lower Fe and Ca’’. Please provide the ‘lower’ numbers, particularly for Fe, since there is no Table for metal content.

Response 5: The authors have included the quantity in the revised manuscript, lines 173-174.

Comment 6: Lines 166-168: ‘’WTWS’. Elsewhere abbreviated as ‘WTS’.

Response 6: The authors thank the reviewer for pointing this out. It was a typo, and authors have made the changes.

Comment 7: Lines 172-173: “The amount of adsorbed contaminant per gram of absorbent sludge with respect to contact time is shown in Figure 1”. It seems that Figure 1 shows the concentration of BTEX in mg/L (starting initially at 45 g/L each) over 30 days.

Response 7: Thank you for highlighting this error. The authors have accordingly modified the sentence. (Revised manuscript lines 177-179)

Comment 8: Lines 174-175: ‘’The concentration of BTEX in the control remained almost constant’’. What is the control made of?

Response 8: The control only contained BTEX containing wastewater which has now been added for clarity on line 184-185 of the revised manuscript.

Comment 9: Lines 225-226: It is stated ‘NWTS exhibited a distinct and a higher lag period before the onset of BTEX degradation and was also the least effective media for degradation of BTEX’’. It does not seem to align with degradation rates presented in Table 1.

Response 9: The authors are thankful to the reviewer for their input and have accordingly made the change in the revised manuscript (line 244-245)

Comment 10: Line 273: the temperature in the lab varied from 1 °C to 24.5 °C? How come?

Response 10: The experiments were started almost when the winter was finishing, hence the low temperatures. There were a few hot days in spring, hence the higher temperatures.

Comment 11: Lines 277-278: It is stated ‘’No correlation was observed between temperature and BTEX degradation’’. It seems unlikely. How was it interpreted?

Response 11: The authors thank the reviewer for their valuable feedback and for highlighting this point. The authors have included the following text in the revised manuscript to support their statement: ‘No clear correlation was observed between temperature and BTEX degradation, but it is likely that rates would be affected by temperature (e.g. Nardi et al., Biodegradation, 2007; Khodaei et al., International Biodeterioration & Biodegradation, 2017). This is an important aspect that future studies should examine.

Comment 12: Section 3.4. Microbial community changes: Too vague. Too long. Not informative enough. What is of special interest are the genera associated with Fe cycling, with aromatic compounds, and some others, but not all. Bacteroides, Firmicutes, and Proteobacteria cover so many different types of microorganisms.

Response 12: The authors thank the reviewers for their critique and have accordingly made the changes.

Comment 13: Keep more emphasis on the genera Gallionella, Rhodoferax, Geobacter, and Geothrix, and aromatic degraders. Is there any relationship in what you observed in the distribution of iron-reducers and iron-oxidizers and the results of the Fe analysis?

Response 13: The authors thank the reviewer and have accordingly made the changes.

Comment 14: Figure 5B. Some OTUs could be regrouped together as ‘others’.

Response 14: The authors thank the reviewer for their comment.

Comment 15: Line 391: it is mentioned ‘WWS was added to enhance the nutritive value of MWTS’ Please include that sentence earlier in the text to explain why you did that mix.

Response 15: The authors have included the following in the revised manuscript line 83: ‘WWS was added to enhance the nutritive value of MWTS.’

Round 2

Reviewer 1 Report

Comments and Suggestions for Authors

It can be accepted in present form.