Ciprofloxacin Removal from Aqueous Media Using Floating Treatment Wetlands Supported by Immobilized Bacteria
and Muhammad Afzal 1,*
Round 1
Reviewer 1 Report
In introduction, other methods of ciprofloxacin removal should be discussed and compared with this manuscript.
If possible, real sample should be applied and evluated using the proposed method.
Determination of ciprofloxacin: In this part, the detection method and principle should be discussed in detail. Such as : provide the "red color complex" chemical structure, and provide possible reaction mechanism between the ferric chloride and the CIP.
Author Response
Comment 1: In introduction, other methods of ciprofloxacin removal should be discussed and compared with this manuscript.
Response:
Other methods of ciprofloxacin removal and their comparisons are now provided in Introduction of the revised manuscript.
“Lines 58-67: Various physicochemical and biological methods such as advanced oxidation, ozonation, reverse osmosis, membrane filtration, electrochemical, and biodegradation have been reported for the removal of antibiotics from water (Del Álamo et al. 2020; Liu et al. 2020; Wang et al. 2021b; Yang et al.2020). However, most of these are expensive and energy/chemical intensive in nature (Rehman et al. 2018; Tara et al. 2019). The use of FTWs, however, is a suitable, economic, and passive treatment alternative that could remove antibiotics from the contaminated water similar to the other contaminants (Benvenuti et al. 2018; Tara et al. 2019; Yasin et al. 2021). This nature-based technology that uses plants and their associated microorganisms to eliminate organic pollutants from contaminated water (Russo et al. 2019; Tara et al. 2019).
Comment 2: If possible, real sample should be applied and evaluated using the proposed method.
Response: We appreciate the reviewer comment. We agree that real sample would have been a best option to study the efficacy of the wetland system; however, we were particularly interested in ciprofloxacin removal at high concentrations (mg/L) whereas real wastewater we tested displayed low concentration of ciprofloxacin (i.e., ng/L to ug/L). Also, the real wastewater had high fraction of other antibiotics as well which could mask the effect of this antibiotic particularly. However, we are now testing the wetland treatment efficiency in this kind of wastewater where we are testing mixture toxicity in the presence of different antibiotics, and results will be published as a separate article.
Comment 3: Determination of ciprofloxacin: In this part, the detection method and principle should be discussed in detail. Such as: provide the "red color complex" chemical structure, and provide possible reaction mechanism between the ferric chloride and the CIP.
Response: We thank the reviewer for this comment. We have now provided detailed methodology and the mechanism in the revised manuscript.
“Lines 191-205: CIP has two functional groups namely an ionizable carboxylic group (pka: 6.05) and a piperanyzyl group (pka: 8.22). The drug show absorption maxima in the visible region at 530 nm. The fluorine atom presents in the quinolones group act as an electron withdrawing group, while the benzene ring in the CIP drug has lower electron density than the terminal nitrogen atom in the piperazinyl moiety. Therefore, CIP serve as n-electron donors to form complexes with metals. This finding supports that the interaction of the CIP drug and reagent takes place at only one site which is the N-atom of piperazinyl ring considering the steric and electron donating factors Akram et al. (2015). Under standard conditions, CIP reacts with ferric chloride to produce a red color complex. This reaction is specific for fluoroquinolones hydrochlorides, which is used for spectrophotometric determination of fluoroquinolones. Briefly, 3 ml aliquot of CIP containing 0.5 to 9.0 μg/ml was added to 3.0 ml of ferric chloride solution. A red color complex is instantly formed which was further diluted in distilled water (10 ml). The complex was then allowed to stand at room temperature (25±5 oC) for half an hour to check its stability. Finally, the absorbance was recorded at 530 nm.
Reviewer 2 Report
Respected authors,
Please find below my comments on the paper:
1- The name or number of the standard methods to measure COD, BOD and TOC should be mentioned in the “Materials and Methods”.
2- Line 147: five g should be changed to 5 g.
3- Line 186: fee should be changed to free.
4- In line 203, what does complete degradation mean? If the degradation is complete and CIP is degraded to minerals, then COD and TOC should reach zero.
5- It is highly recommended to identify the byproduct of CIP degradation.
6- Authors should justify achieving lower TOC compared to COD after 20 days for the T4 sample. If COD is high, it means more organic compounds exist in the solution, while this is not supported by the TOC results.
7- All results are displayed using bar graphs. It would be more appealing to use other styles of results presentation.
Yours sincerely,
Author Response
Comment 1: The name or number of the standard methods to measure COD, BOD and TOC should be mentioned in the “Material and Methods”.
Response: Thank you for the careful review and suggestion. We have now added the suggested information in the revised manuscript.
“Lines 182-185: APHA standard methods were used measure water quality parameters such as pH (method 4500-H), EC (method 2510), turbidity (method 2130), Chemical oxygen demand (method 5220), biochemical oxygen demand (method 5210B), and total organic carbon (method 5310) (APHA, 2005).
Comment 2: Five g should be changed to 5 g.
Response: Thank you for the comment. We have now fixed it in the revised manuscript.
“Lines 182-183: The suspension was prepared by using a pestle and mortar to ground 5 g of the roots and shoots in the normal saline (0.9%, w/v).
Comment 3: Fee should be changed to free.
Response: Thank you for the careful review. Now, it has been updated in the revised manuscript.
“Lines 264-266: “More CIP removal was observed in the treatments comprising immobilized cells than the treatments having free bacteria.”
Comment 4: In line 203 what does complete degradation mean? If the degradation is complete and CIP is degraded to minerals, then COD and TOC should reach zero.
Response: Thank you for the comment. In principle, CIP mineralization does not necessarily mean 100% removal of COD and/or TOC rather intermediates (transformation products) such as smaller organic compounds could be formed. Often, these organics are further degraded by the microbial communities over time. A smaller fraction is also expected to be taken up by plants (roots and shoots). Ideally, a detailed analyses on CIP byproducts could have explained the degradation mechanisms, and answered this question specifically, but due to technical limitations, we could not perform the analysis. However, these analyses are expected to be performed in our future studies.
Comment 5: It is highly recommended to identify the byproduct of CIP degradation.
Response: Thank you for the suggestion. As mentioned above, due to technical limitations we could not study the byproduct of CIP degradation; which requires molecular work and was also the beyond the scope of the study. We are however planning to do this analysis in our future studies. But speculated interpretation on byproduct identification is provided in the revised manuscript.
“Lines 330-334: The biodegradation of CIP could have occurred by the cleavage of isoxazole and piperazinyl rings via sulfite reductase and cytochrome P450 (CYP450) enzymes, respectively (Jia et al. 2018). A previous study has also shown that 100% removal of CIP may be achieved by effective microbial degradation (Li et al. 2021).
Comment 6: Authors should justify achieving lower TOC compared to COD after 20 days for the T4 sample. If COD is high, it means more organic compounds exist in the solution, while this is not supported by TOC results.
Response: Thanks for the comment. Principally, a high COD could also be due to the presence of inorganic reducing substances, which are less in the case of TOC. Further, TOC reflects dissolved organic fraction and the water is pre-filtered for any suspended particles; however, for COD, we used raw wastewater. So, we request reviewers to consider results from both experiments independently.
Comment 7: All results are displayed using bar graphs. It would be more appealing to use other styles of results presentation.
Response: Thanks for the suggestion. For consistency and better comparisons, we used bar graphs throughout the manuscript. We hope that reviewer would agree to our comment.
Reviewer 3 Report
Comments for author File: 
Comments.pdf
Author Response
Reviewer: 3
The manuscript entitled “Ciprofloxacin removal from aqueous media using floating treatment wetlands supported by immobilized bacteria” deals with the removal of Ciprofloxacin (CIP) from aqueous solution using floating treatment wetlands supported by immobilized bacteria Phragmites australis. The manuscript was submitted by Syed Wajid Ali Shah, Mujaddad ur Rehman, Muhammad Tauseef, Ejazul Islam, Azam Hayat, Samina Iqbal, Muhammad Arslan, and Muhammad Afzal can be considered for publication in Sustainability Journal after a serious major revision.
Response: Thank you for the comments. We have now revised the manuscript considering comments of all three reviewers. We believe that these comments helped us address the weak aspects of the manuscript and now, the readability is improved significantly. We appreciate the reviewer’ critical feedback on our manuscript. The responses can be seen below.
Comment 1: The utility of this study should be highlighted in the manuscript and explain the real-time application of immobilized bacteria Phragmites australis concerning a large scale.
Response: Thank you for the suggestion. We agree! More information on scalability and real-time application is now discussed in the revised manuscript.
“Lines 71-78:
“Practical applications: This study confirms that attached growth system can help achieve maximum remediation in a short time period. This is mainly due to low shear force and better biofilms formation on the fixed bed which is generally followed by effective catabolic activities by the inoculated microbial communities. Arslan et al., (2022) has recently argued that remediation efficiency can be significantly enhanced in the presence of attached-growth systems as compared to the suspended growth systems. Also, attached growth system support high biomass formation, retaining extracellular polymeric substances, with a rich microbial community to drive pollutant transformation effectively. Compared to the suspended biological treatment processes, immobilized and fixed bed treatment systems also require less aeration without any need of additional organic carbon addition. The high biomass can further enhance flocculation and sedimentation of suspended particles in highly turbid wastewater often present in the field-based systems. The overall approach appears to be scalable, sustainable, and cost-effective as observed in our earlier field-scale floating wetlands studies (Afzal et al., 2019).
Comment 2: The innovation and the significance of this work are not clearly highlighted in the abstract, and introduction. Please work on this and prove to us why this work is valuable. What was the optimum concentration of Ciprofloxacin in the wastewater? (as per pollution control voard standard value).
Response: Thanks for the comment. We have now provided more information to enhance the study novelty at the end of the introduction.
Lines 96-106: The concentration of CIP in Pakistani streams is found in the range of 42-332 μg mL-1 followed by Ofloxacin > Ampicillin > Levofloxacin > Sulfamethoxazole (Zafar et al. 2020). River Ravi in Pakistan is already recognized as a highly pollutant river in the world due to pharmaceuticals (Wilkinson et al., 2022); nevertheless, no current treatment infra-structure exists in the country which signifies the importance of cost-effective and scalable treatment solutions in the country. Previously, it was argued that antimicrobials may negatively affect in planta bacterial communities in wetlands (Arslan, 2015); therefore, scope of alternative approaches considering microbial colonization in fixed beds or via immobilization strategies should be investigated. We further used locally available wetland plant, Phragmites australis, which is a helophytic grass and known to withstand harsh environmental conditions (Saleem et al., 2019). The species also allows adsorption of organics on to root surface, without being translocated to aboveground plant parts, rendering it suitable for the subsequent degradation activities of immobilized bacteria. Further, helophytic grasses can transport atmospheric oxygen into the rhizosphere which help create redox zone around the root for efficient pollutant mineralization (Syranidou et al., 2016). This study hypothesizes that bacterial immobilization on floating raft could be a useful strategy to treat high concentration pharmaceutical contaminated wastewaters.
Comment 3: The rationale why the Phragmites australis bacteria was selected for this study? On what basis did the authors select Ciprofloxacin for the study? The author should explain the adverse effect of Ciprofloxacin in the introduction section.
Response: Thanks for the comment. We have now provided reasons for the use of Phragmites, CIP, and adverse effects of CIP, in the introduction.
Lines 111-120: We used an indigenous wetland plant, Phragmites australis, which is a helophytic grass and known to withstand harsh environmental conditions of Pakistan (Saleem et al., 2019). The species also allows adsorption of organics on to root surface, without being translocated to aboveground plant parts, allowing organics to be available for degradation by immobilized bacteria. Further, helophytic grasses can transport atmospheric oxygen into the rhizosphere which help create redox cone in the rhizosphere (Syranidou et al., 2016). This study hypothesizes that bacterial immobilization on floating raft could be a useful strategy to treat high concentration pharmaceutical contaminated wastewaters.
Lines 103-105: The concentration of CIP in Pakistani streams is found in the range of 42-332 μg mL-1 followed by ofloxacin > ampicillin > levofloxacin > sulfamethoxazole (Zafar et al. 2020).
“Lines 55-63: The presence of antibiotics and/or their residues in the environment is of concern due to non-target toxicity. Precisely, it can alter the functioning of basic nutrient cycles (e.g., carbon, nitrogen, and oxygen) after disturbing the microbial community structures in the particular (micro)ecosystem. Resultantly, unwanted occurrence of antibiotics can affect the biogeochemical cycling of the nutrients driven by indigenous microbial activities. Fi-nally, most susceptible members of the bacterial community are eliminated/inhibited while other members may get a competitive advantage and their abundance may in-crease, cf. concept of intrinsic resistance (Arslan, 2019). Additionally, fluoroquinolones could also cause physiologically teratogenic and genotoxic effects in living organisms.
Comment 4: Why does the author prefer the floating treatment wetlands method? On what basis author not considered other matrices for immobilization? Why was the author not considered for the fungal treatment?
Response: We appreciate reviewer concerns. Indeed, other treatment methods could be tested but our group is largely working on optimization of bacterially-assisted floating wetlands in Pakistan, from lab-scale to field scale. We have seen best results using these approaches so far and we aim to focus on similar research themes in future as well. Our earlier studies have addressed large-scale wastewater treatment via floating wetlands and the results were published in top notch journals including Nature Sustainability. Some examples can be seen below:
https://www.nature.com/articles/s41893-019-0350-y
https://www.nature.com/articles/s41545-018-0025-7
https://sustainabilitycommunity.springernature.com/posts/52081-floating-wetlands-for-low-cost-wastewater-treatment
https://www.nature.com/articles/s41598-022-08517-1
Further, density of bacteria on the substrate material depends on the structure, pore size, and surface area of the support, as well as the nature of the material (hydrophobicity, charge, etc.) and environmental conditions, such as ionic strength, presence of trace elements, pH, and temperature. The bioremediation rate can be enhanced using immobilization methods, particularly under harsh conditions; therefore, in this study we used polystyrene sheet as a carrier for bacterial immobilization. More description is now added in the revised manuscript.
Comment 5: In batch studies, why the Design of experiments (optimization) studies was not included? The optimization of various process parameters using response surface methodology (RSM) should be added to increase the quality of the paper. Also, interpret the data in the results and discussion section. The author should explain the effect of various parameters on the removal of Ciprofloxacin from contaminated water.
Response: Thanks for the critical comments of the reviewer. We have already performed RSM study for CIP removal in batch experiments. The results are already published in ACS Omega journal. Based on these results, we designed the floating wetland study.
https://pubs.acs.org/doi/full/10.1021/acsomega.2c02448
Shah, S.W.A., Rehman, M.U., Arslan, M., Abbasi, S.A., Hayat, A., Anwar, S., Iqbal, S. and Afzal, M., 2022. Response Surface Methodology for Optimization of Operational Parameters To Remove Ciprofloxacin from Contaminated Water in the Presence of a Bacterial Consortium. ACS omega, 7(31), pp.27450-27457.
We have now revised the discussion as well. However, we avoided unnecessary speculation as much as we could, given that we already have added some related discussion in the manuscript. Discussing effect of various parameters on the removal of Ciprofloxacin from contaminated water would be beyond the scope of study and would be too much speculation. We hope reviewer would agree with our responses.
Comment 6: The author should include the kinetic studies for the removal of Ciprofloxacin from aqueous solution and analyze the kinetic data in the results and discussion section. Why the kinetic studies were not conducted?
Response: Thanks for the comment. We agree that kinetics study would have been beneficial but we don’t have enough number of sampling events (n=3) to produce valid conclusions. Applying kinetics on less time points may lead to false conclusions, which we don’t want to do. Therefore, this was not an option for this study, as well as it was beyond the study scope. However, we will try to include kinetics work in our future studies.
Comment 7: The author should specify the details to buy the bacterial culture in line no. 90
Response: Thanks for the comment. The bacterial strains used for consortium was previously isolated and characterized in Soil and Environmental Biotechnology Divisions. We did not buy bacterial culture. The related text can be seen below, which is provided in the manuscript.
Lines 134-139: A consortium consisting of three bacterial strains, A. lwoffiii (Fatima et al. 2015), B. pumilus (Anwar et al. 2009), and Mesorihizobium sp. (Jabeen et al. 2015), was used in the current study. The strain A. lwoffii was previously isolated from the rhizosphere of Acacia ampliceps, whereas B. pumulis and Mesorihizobium sp. were isolated from pesti-cide-contaminated soil. These strains and their consortium showed in vitro potential to degrade CIP (100 mg L-1) in a minimal salt medium.
Comment 8: The author should explain, how to measure the concentration of Ciprofloxacin remaining in the contaminated water and incorporate the details in the manuscript. The author should explain to evaluate the toxicity level.
Response: Thanks for the comment. We have added the detail methodology to measure CIP concentration and toxicity in the revised manuscript.
“Lines 214-221: Under standard conditions, CIP reacts with ferric chloride to produce a red color complex. This reaction is specific for fluoroquinolones hydrochlorides, which is used for spectrophotometric determination of fluoroquinolones. Briefly, 3 ml aliquot of CIP containing 0.5 to 9.0 μg/ml was added to 3.0 ml of ferric chloride solution. A red color complex is instantly formed which was further diluted in distilled water (10 ml). The complex was then al-lowed to stand at room temperature (25±0.5 oC) for half an hour to check its stability. Finally, the absorbance was recorded at 530 nm.
“Lines 246-253: The toxicity of raw and treated wastewater was studied in-vitro using wheat (Triticum aestivum L.) seeds (Rehman et al. 2019). Briefly, five seeds were put in a dish having filter paper saturated with distilled water, or treated and untreated CIP-contaminated water. Seeds were allowed to germinate at room temperature (25 °C) in darkness. The test was conducted in triplets. After five days, germinated seeds were viewed. Seeds were be-lieved to have germinated when the combined length of the plumule and radicle was more than 2 mm. Plantlets growth (root length and total length) was measured.
Comment 9: The author should incorporate the analysis of enzyme activity for the removal of
Ciprofloxacin.
Response: We appreciate the reviewer comment. The main scope of the study was to study the efficacy of FTW for CIP removal, and if the results can be translated in the field (ongoing phase). The enzyme activity was therefore not the main focus and therefore, we are not able to provide these details unfortunately. However, we will consider these analyses in our future studies but a relevant and speculated interpretation on biodegradation mechanism in the revised manuscript.
“Lines 338-340: The biodegradation of CIP could have occurred by the cleavage of isoxazole and pi-perazinyl rings via sulfite reductase and cytochrome P450 (CYP450) enzymes, respec-tively (Jia et al. 2018).”
Comment 10: I recommend adding comparisons of specific experimental data obtained with the results of the authors of other works in the results and discussion section. An author should add a high-resolution graphical image. Also, English needs to be carefully checked again and polished.
Response: Thank you for the comments. We have now added necessary comparisons in the revised manuscript, considering comments of other two reviewers as well. English is proofread senior authors (Dr. Muhammad Arslan, University of Alberta & Dr. Muhammad Afzal, NIBGE).
Comment 11: Authors should rewrite the conclusion part, it should be more sound with significant results.
Response: thanks for the comment. Now the conclusion part is updated accordingly.
Lines 460-470: The application of immobilized bacteria onto polystyrene sheet significantly in-creased CIP remediation (97%) via FTW. To this end, helophytic grasses such as Phragmites australis could be a suitable option due to its metabolic abilities to support microbial proliferation and enhance organics mineralization in the root zone (German: Wurzelraumverfahren). The bacterial strains A. lwoffii ACRH76, B. pumulis C2A1, and Acinetobacter sp. HN3 were able to develop successful partnership with the plant, even in the presence of CIP, indicating abilities to survive in antibiotics stress. FTW supported by immobilized bacteria offers sustainable and scalable solutions to treat wastewater contaminated with antibiotics. Owing to near-natural means of remediation and comparable little energy requirements, the technology is particularly attractive for countries with low economic constraints such as Pakistan. Further studies are, however, needed to explore the metabolic activity of the bacteria during the degradation of CIP.
Comment 12: The authors must delete the old references and should include the new references (After 2018). So that the research in this field was updated.
Response: We appreciate the reviewer’s comment. We have updated the reference list as per the reviewer comments. However, at some places, we could not delete old references considering that they are original source of the information.
Round 2
Reviewer 2 Report
Respected authors,
Please find below my comments on the paper:
1- For the complete degradation of CIP, authors must state the formation of new byproducts and the fact that many of them can be even more toxic than the original CIP. Mentioning only the complete degradation of CIP is misleading.
2- I am still not convinced that showing the results only in the style of bar graphs helps with consistency and better comparison. Then why in different studies, the researchers are trying to be more creative in representing their results?
Yours sincerely,
Author Response
Dear Reviewer,
We appreciate your comments. Here is our further response to remaining two questions you have highlighted!
Comment 1: For the complete degradation of CIP, authors must state the formation of new byproducts and the fact that many of them can be even more toxic than the original CIP. Mentioning only the complete degradation of CIP is misleading.
Response: We already conducted seed germination toxicity bioassay to investigate if treated water was toxic after treatment (due to intermediates). Our results indicated that treated water in the presence of plants and bacteria was non-toxic while the results were comparable to the plants grown in tap water. This indirectly confirms that the wastewater could be re-used for agricultural purposes, a proxy parameter. The study of byproducts would not possible at this time as it requires in depth investigations via HPLC. We are not able to report these findings within the timelines of this review process (10 days). However, wehave replaced the word "complete" with "efficient". We also believe that metabolite study would be a completely new study and is currently beyond the scope of this study. Here, you can see a similar previous work we conducted to study metabolites recently.
https://link.springer.com/article/10.1007/s11356-021-15130-8
2- I am still not convinced that showing the results only in the style of bar graphs helps with consistency and better comparison. Then why in different studies, the researchers are trying to be more creative in representing their results?
Response: With due respects, we would like to argue that different styles of plotting is just a matter of taste. However, considering reviewer's comment, and broad readership of the journal, we have now provided some results as boxplots in the revised manuscript. We hope this would suffice the need of review purpose.
Round 3
Reviewer 2 Report
The authors have addressed the comments appropriately.
The paper can be accepted for publication.
Regards,
