Plant-Microbe Synergism in Floating Treatment Wetlands for the Enhanced Removal of Sodium Dodecyl Sulphate from Water

Round 1

Reviewer 1 Report

The research paper entitled "Plant-Microbe Synergism in Floating Treatment Wetlands for the Enhanced Removal of Sodium Dodecyl Sulphate from Water" by Afzal  et al. is interesting and important for sustainable development in environmental concern.

However, many points are not clear that must be improved before reconsideration.

The authors have to address the following points:

1. what are TYRH47 and BRSI56 in Abstract. The abbreviations are needed.

2. Introduction of ionic surfactants is lack of application and the reason for removal.
   The authors should refer some papers:
   Journal of Molecular Liquids 287, 110900 (2019); Colloid and Polymer Science 293 (1), 217-227 (2015); Environmental Chemistry 14 (5),
   327-337 (2017); https://doi.org/10.1155/2020/6676320; https://doi.org/10.1002/elps.202000086

3. The reason for selection of SDS as an anionic surfactant should be emphasized.
4. Detailed analytical method of SDS by spectrophotometry should be added.
5. Discussion on the results of SDS removal is week. Only 13 % removal of SDS is too low.
6. Discussion on the results in Figures 2-4 are not clear.
7. Discussion on persistence of Inoculated bateria is also week.
8. Heading of Table 2 and its content should be in one page.
9. Conclusions should be rewritten to again the new findings in the present study.

Author Response

The research paper entitled "Plant-Microbe Synergism in Floating Treatment Wetlands for the Enhanced Removal of Sodium Dodecyl Sulphate from Water" by Afzal et al. is interesting and important for sustainable development in environmental concern.

However, many points are not clear that must be improved before reconsideration.

The authors have to address the following points:

Response: We thank reviewer #1 for the positive comments. We have now revised the manuscript carefully considering comments and suggestions of both reviewers. We believe that the manuscript is now significantly improved and hopefully reviewers would agree to it as well. Thanks!

1) what are TYRH47 and BRSI56 in Abstract. The abbreviations are needed.

Response: TYRH47 and BRSI56 are strains identifiers based on their IGS type (intergenic spacer region). These strains were previously isolated by Fatima et al., 2015.

• Kaneez, F., Muhammad, A., Asma, I. and Khan, Q.M., 2015. Bacterial rhizosphere and endosphere populations associated with grasses and trees to be used for phytoremediation of crude oil contaminated soil. Bulletin of Environmental Contamination and Toxicology, 94(3), pp.314-320.

Their sequences have been submitted to NCBI including strain information (TYRH47 and BRSI56). Therefore, for the sake of accuracy, we added the exact information in the revised manuscript. We would also like to mention that the same writing method is used previously in many articles. Here are some examples:

• Tara, N., Arslan, M., Hussain, Z., Iqbal, M., Khan, Q.M. and Afzal, M., 2019. On-site performance of floating treatment wetland macrocosms augmented with dye-degrading bacteria for the remediation of textile industry wastewater. Journal of cleaner production, 217, pp.541-548.
• Hussain, Z., Arslan, M., Malik, M.H., Mohsin, M., Iqbal, S. and Afzal, M., 2018. Treatment of the textile industry effluent in a pilot-scale vertical flow constructed wetland system augmented with bacterial endophytes. Science Of The Total Environment, 645, pp.966-973.

2) Introduction of ionic surfactants is lack of application and the reason for removal. The authors should refer some papers: Journal of Molecular Liquids 287, 110900 (2019); Colloid and Polymer Science 293 (1), 217-227 (2015); Environmental Chemistry 14 (5), 327-337 (2017); https://doi.org/10.1155/2020/6676320; https://doi.org/10.1002/elps.202000086

Response: Thank you for the comment. We have now added the suggested references and also included the information on type of surfactants with a focus on anionic surfactants. The revised text reads as follows:

“Further, they are used to modify many adsorbents to increase the efficiency of removing many pollutants. Traditionally, surfactants have been categorized into four types based on the hydrophilic heads, namely, anionic, cationic, amphoteric, and nonionic [16]. Among them, ionic surfactants have received tremendous attention because they are very popular, strongest and most inexpensive agents [17-18]. They have a negative charge on their hydrophilic end that helps the surfactant molecules lift and suspend particles in micelles. In the micelle, the surfactants are oriented with their charged head groups toward the solid surface while the hydrophobic hydrocarbon chains protrude into aqueous phase followed by their effective removal from the contaminated environment [18-19]”

3) The reason for selection of SDS as an anionic surfactant should be emphasized.

Response: Thank you for the comment. We have now added the following text:

“We selected SDS as a model compound because it is abundantly found in the domestic wastewater. It has extensive application in cleaning and hygiene products such as household detergents, car wash shampoos, soaps, and toothpaste [15]. The adsorption potential of SDS on various substrates is already investigated; nevertheless, removal efficiency by CWs from the contaminated wastewater is still unknown. In this study, we investigated the treatment performance of the system by evaluating temporal decrease in SDS concentration, COD, and BOD reduction in the water.”

4) Detailed analytical method of SDS by spectrophotometry should be added.

Response: We thank you the reviewer for the careful review. We have now added the following text in the revised manuscript.

“At first, 1 drop of 1% phenolphthalein solution was added to the solution as an indicator. Then, 1 M NaOH was added until the color was changed to pink, which was followed by the addition of 1 M H2SO4 until the solution became colorless. The chloroform and methylene blue reagents were then added in the solution. All of the procedure was done in a separatory funnel. The flasks were shaken for about 30 seconds and for the phase separation, these flasks were left for 30 minutes. The chloroform layer was extracted in a 100 ml of Erlenmeyer flask. The procedure was repeated thrice by adding 5 ml of chloroform. Three layers of chloroform were obtained. The chloroform layer was extracted in a volumetric flask. The absorbance of chloroform was measured by a spectrophotometer at 652 nm against the blank chloroform. The blank was prepared by adding 5 ml phosphate buffer solution, 2 ml cationic dye (methylene blue) solution, and 5 ml of ex-tracting solvent (chloroform) in100 ml distilled water. Standard solution was prepared by adding 5 ml phosphate buffer solution, 2 ml the dye solution, and 5 ml of extracting solvent in 100 ml of 10 ppm standard SDS solution. The calculations were performed as follows:

Standard Factor= 10/OD of standard solution

Detergents (mg/L) in wastewater sample = OD of sample x SF”

5) Discussion on the results of SDS removal is week. Only 13 % removal of SDS is too low.

Response: Thank you for the comment. In this study, 13% removal of SDS was observed in the control where no vegetation and bacterial consortium was present. However, in the presence of vegetation and bacteria, the removal of SDS was significantly enhanced. This has now been clearly mentioned in the revised manuscript.

“In this study, 13% removal of SDS was also observed in the control when no vegetation and bacterial consortium was present. This could be attributed to natural factors such as photooxidation, adsorption, and indigenous role of microbes. However, the degradation was further enhanced by vegetation and bacterial inoculation. Specifically, SDS removal was enhanced to >90% when plants and bacterial consortium was applied together. Nevertheless, performance of B. mutica and bacterial consortium was best be-cause ~98% removal was achieved in this treatment in a period of 5 days.”

6) Discussion on the results in Figures 2-4 are not clear.

Response: We thank the reviewer for a careful review. We have now revised the whole discussion for the figures 2-4 carefully. The following paragraphs has been added to improve the discussion further.

“Usually, plants can take up the organic contaminants from the environment if the water octanol partition coefficient (log kow) ranges between 0.5 to 3.5. they cross the cell membrane. The log kow values of SDS is 1.6 that makes it an easy compound to be taken up by the plants. This might be the reason that even vegetation alone significantly reduced the SDS concentration and other pollution parameters in the contaminated water. Nevertheless, performance of B. mutica was better than L. fusca which could be due to the better adaptability of B. mutica in this kind of wastewater. It is previously reported that B. mutica outperforms in the wetlands even under harsh environmental conditions for the removal of variety of organic and inorganic contaminants [41].”

“This could be due to the effective plant-microbe interplay in the FTWs: (1) inoculated bacteria were previously isolated from the shoot and root interior of plants so they could have already developed mechanisms of proliferation in the plant rhizo- and endosphere that allow the bacteria have helped degrade the SDS and supported the health of host plant in a synergistic manner, and (2) the bacteria possessed genes involved in pollutant degradation and plant growth promoting activities, i.e., 1-aminocyclopropane-1-carboxylate (ACC) deaminase, siderophores production, phosphorus solubilization [35-36, 41-42, 57].”

“In our earlier investigations, we found that B. mutica allows proliferation of diverse and rich bacterial in the rhizo- and endosphere that overall improves the health of the host plant and degradation potential of the wetland system [42].”

“Both COD and BOD are important water quality parameters and their attenuation indicates the cleaning of contaminated water [42]. Alongside, high oxygen concentration is fundamental to such environment and successful interactions among plant roots and associated bacteria rely on the availability of oxygen diffusion. In a well growing FTW system, vegetation could have provided oxygen in the rhizosphere through the plant roots thus allowing the microbes to nurture and ultimately leads to the degradation of contaminants [42, 54, 55].”

7) Discussion on persistence of Inoculated bateria is also week.

Response: Thank you for the comment. We have now revised the discussion on persistence of the inoculated bacteria. The following information is added now:

“The survival and colonization of inoculated bacteria in the FTWs is highly crucial for efficient degradation of the contaminants [42]. In this study, inoculated bacteria showed survival in the root and shoot interiors of B. mutica and L. fusca. This could be due to the fact that all of the inoculated bacteria were previously isolated from the rhizosphere, roots, and shoots of the wetland plants; therefore, they could have developed necessary mechanisms to colonize the plant interior for B. mutica and L. fusca as well [35-36, 41-42, 57]. Further, we observed that the bacterial population in the root interior was significantly higher than that of the shoot interior of both plants. The higher population in the roots could be attributed to the fact the inoculated strains were often observed in the rhizospheric and root interior of the wetland plants in earlier studies, which suggest their better colonization potential in the root environment compared to the shoot [52]. Also, in this study, the observations were made after a few days only and the time might not have been sufficient for the active migration of the bacterial communities to the aboveground tissues.”

8) Heading of Table 2 and its content should be in one page.

Response: Thank you for the comment. This has been fixed now.

9) Conclusions should be rewritten to again the new findings in the present study.

Response: We have now completely revised the conclusions as per our best understandings. The new conclusions read as follows:

This study establishes the usefulness of exploiting rhizospheric and endophytic bacteria in FTW in a partnership with two wetland plants namely B. mutica and L. fusca for reclamation of water contaminated with SDS. We argue that a traditional FTW can be an effective choice for enhanced SDS removal from the wastewater if well-screened bacterial communities are inoculated in the system. In this way, a successful attenuation in COD, BOD, and pollutant of interest (SDS) could be achieved in a very short time. This study also argues that, if inoculated bacteria are compatible with the host and do not compete for resources with each other, they can survive well in planta, support the host health, and improve pollutant degradation. The better performance of B. mutica nevertheless indicates that different plants have different capacity of effective plant-microbe interplay which should be investigated carefully before designing an experiment. In the end, this study strengthens the application of pollutant degrading bacteria in FTW for the remediation of water contaminated with organic compounds. Nevertheless, further studies on the activity of enzymes alkyl sulfatases for the degradation of SDS are suggested.

Author Response File: Author Response.pdf

Reviewer 2 Report

I don´t have any other comments

Author Response

We thank you the 2nd reviewer for the positive comments. We have improved the manuscript further as per the comments of 1st reviewer, and we feel that you will also feel that it is significantly improved. 

Thanks for your time and support in publishing this article.

Thanks

Round 2

Reviewer 1 Report

The revised paper was significantly improved by the athors.

The responses to reviewers'comments are suitable.

The manuscript can be accepted in current form.

Author Response

We appreciate the time and effort reviewer has put in improving our manuscript. We are also thankful for the acceptance of our manuscript in Sustainability. 
Best regards