Adsorption Behavior of Inorganic and Organic Phosphate by Iron Manganese Plaques on Reed Roots in Wetlands
Round 1
Reviewer 1 Report
Comments to the manuscript: Adsorption Behavior of Phosphorus by Iron-manganese Plaque on Reed Roots in Wetland
The manuscript deals with the interesting issue of removing organic and inorganic phosphorus by Fe-Mn plaque.
Comments to the manuscript:
There is no clearly marked the aim of the study! Please indicate why You did the research.
Material and methods
This part have to strictly rewrite. The parts of methodology are mixed with Results and Discussion part!
2.3 the subsection is very general, please detail it.
Some parts of methodology seems to be an aims of the study… l. 82-83
2.4-2.6 How many repetitions you did?
l. 96 designed time intervals – please specified
Results and Discussion
Fig 1. What is the scale of a and b? It seems to be different and maybe that the reason of differences.
l.142-143 this is not a part of results and discussion . It more lake a aim… Please rewrite it.
l.185-192 This a methodology!
l.205-212 This a methodology!
l.231-241 This a methodology!
Generally, there is lack of discussion of obtained results. Authors only focused on results. Please improve it.
Please add the part of adsorption mechanism.
What is the utilization part of yours study?
l.174-175 this is a repetition.
Table 3. FMO – what is this abbreviation?
Table 4 instead Table 3 (is doubled)
l. 249-251 – repertion , that data is presented in Table 4. Please change it
Conclusion
There were no mention of chemisorption and monolayer adsorption in the Results and Discussion part…
Generally, the manuscript is very chaotic and have to be strictly rewritten.
Author Response
Response to Reviewer 1 Comments
Point 1
There is no clearly marked the aim of the study! Please indicate why You did the research.
Response1 : Thanks for the reviewer’s suggestion.
In China, phosphorus residues in wastewater treatment plants effluent are generally removed primarily using a wetland system before the effluent is discharged into a water body. In such wetland systems, phosphorus is removed by plant uptake, media adsorption and microorganism assimilation. Media adsorption is usually limited because much of the solid matter is inert and has a poor adsorption capacity. Assimilation by microorganisms contributes little to the removal of total phosphorus because the phosphorus will be released back to the water once the microorganisms die. Plant uptake is normally considered to affect phosphorus removal little until the plants are harvested. However, oxygen can be released from the dense root networks of plants in wetlands, creating an oxidizing rhizosphere environment. This can cause Fe2+ and Mn2+ in the rhizosphere soil or media to become oxidized to Fe3+ and Mn4+, respectively. The Fe3+ and Mn4+ can precipitate and accumulate on the root surfaces, to form features called iron manganese (Fe-Mn) plaques. Fe-Mn plaques on hydrophyte root surfaces can immobilize various nutrients and metalloids. Wang et al. found Arundo donax Linn and Typha latifolia roots had higher phosphorus contents when iron plaques were presented than when iron plaques were not present. Chong et al. found that amorphous iron oxide on root surfaces caused phosphorus to accumulate in the rhizosphere.
It has been shown the phosphorus adsorption behaviors and mechanisms by Fe-Mn plaques are not explained clearly, and the influence between inorganic and organic phosphate is ignored. Therefore, to offset the disadvantages of other phosphorus removal ways, the aim of our study is to figure out the contribution of inorganic and organic phosphates removal by Fe-Mn plaque, and provide guidance for the application of this long-term effective and sustainable phosphorus removal through controlling the interior habitat to promote the formation of Fe-Mn plaque in waterfront wetland systems.
The aim of the study has been specifically indicated in the line 55-58 in Introduction chapter in the revised manuscript.
Point 2
Materials and Methods
This part have to strictly rewrite. The parts of methodology are mixed with Results and Discussion part!
Response 2: Thanks for the reviewer’s constructive comment. The Materials and Methods chapter has been strictly rewritten as follows:
2. Materials and Methods
2.1 Chemicals
All chemicals were of analytical reagent grade and were purchased from Beijing Chemical Co. (Beijing, China). Solutions of inorganic and organic phosphates for use in the adsorption tests were prepared by dissolving potassium dihydrogen phosphate (KH2PO4) and adenosine-5’-monophosphate (C10H14N5O7P), respectively, in ultrapure water. The ultrapure water (18.2 ΩM·cm-1) used throughout the study was prepared using a Millipore system (Merck, Darmstadt, Germany).
2.2 Fe-Mn Plaque Collection
The rReeds with roots were collected from a wetland at the Beijing University of Civil Engineering and Architecture (Beijing, China). The wetland is used to treat effluent from a sewage treatment plant. The roots were cut from the reeds and washed thoroughly with ultrapure water. The roots were then placed in a beaker containing ultrapure water and ultrasonicated for 5h using a KQ-500B instrument (Kun Shan Ultrasonic Instruments Co., Ltd, Kun Shan, China). This caused the Fe-Mn plaques to separate from the root surfaces and become suspended in the water. The process was repeated until sufficient suspension was obtained to perform the planned tests. The suspended plaques were reddish brown. The suspension was evaporated and freeze dried to give dry Fe-Mn powder, which was stored in a desiccator. The iron and manganese contents of the Fe-Mn plaque powder were determined by acid digesting and then analyzing the solution using a Hitachi Z-2010 atomic absorption spectrometer (Hitachi High-Technologies, Tokyo, Japan). The total phosphorus and inorganic phosphorus contents of the Fe-Mn plaque powder were determined and used as the background concentrations (i.e., before adsorption experiments were performed). The weights of the roots were determined before the ultrasonic extraction process was performed.
2.3 Characterization
The reed roots with and without Fe-Mn plaques attached were examined by SEM using an S-3500N instrument (Hitachi High-Technologies). Before being examined, the samples were sputtering coated with gold and palladium for 45 s using a Quorum Polaron SC7620 mini-sputter coater (Quorum Technologies Ltd, East Sussex, UK) to decrease charging effects inside the microscope [16].
The electron binding energies and the oxidation states of iron and manganese in the plaques were determined by XPS using a Shimadzu ESCA-lab-220i-XL instrument (Shimadzu, Kyoto, Japan) using monochromatized Alkα X-rays at 1486.4 eV.
Freeze dried Fe-Mn plaque samples before and after inorganic and organic phosphate had been adsorbed by plaques were analyzed by FTIR. Each sample was mixed with spectral grade KBr at a weight ratio of 100:1 and pressed to form a disk. The disks were analyzed using a Nicolet 6700 FTIR spectrometer (Thermo Fisher Scientific, Waltham, MA, USA). FTIR spectra over the range 4000 - 500 cm-1 were acquired and the functional groups in the samples were identified.
2.4 Adsorption Kinetics
kinetics of inorganic and organic phosphate adsorption by the Fe-Mn plaque powder. 0.1 g Fe-Mn plaque powder was added to 200 mL solutions containing 6 mg/L inorganic or organic phosphate at pH 7.0 in a 250 ml conical flask. The conical flasks were agitated at 120-130 rpm using a mechanical orbital shaker at 298 K. An aliquot of the solution in each test was sampled at each specified time intervals (5min, 10min, 20min, 30min, 1h, 2h, 4h, 8h, 16h and 24h) and passed through a 0.45 μm polycarbonate membrane filter, then the inorganic phosphorus concentration was determined using an ammonium molybdate spectrophotometric method and the total phosphorus concentration was determined using an alkaline potassium persulfate digestion spectrophotometric method, using a Sunny Hengping Instrument 752 ultraviolet visible spectrophotometer (Sunny Hengping Instrument, Shanghai, China) at wavelength of 700 nm. The organic phosphorus concentration was defined as the difference between the total phosphorus and inorganic phosphorus.
The mechanisms and steps controlling adsorption were analyzed by fitting the pseudo-first-order and pseudo-second-order models expressed in the Equations (1) and (2) [17,18], respectively, to the kinetics data.
[please see the revised manuscript]
Where t (min) is the contact time, Qe (mg/g) and Qt (mg/g) are the amounts of phosphate adsorbed at equilibrium time and time t, and K1 (1/min) and K2 (g/mg/min) are the pseudo-first-order and pseudo-second-order adsorption rate constants, respectively.
2.5 Adsorption Isotherms
Tests were performed to investigate the isotherms for the inorganic and organic phosphate adsorption by the Fe-Mn plaque powder. In each test, 0.1 g Fe-Mn plaque powder was added to 200 mL solution containing inorganic or organic phosphate in a 250 mL conical flask. Tests were performed using initial phosphorus concentrations between 0.1 mg/L and 6 mg/L at pH 7.0. The conical flasks were agitated at 120-130 rpm using a mechanical orbital shaker at 298 K. After 24 h, each solution was passed through a 0.45 μm polycarbonate membrane filter and the residual inorganic and organic phosphorus concentrations were determined.
The Langmuir and Freundlich models, shown in Equations (3) and (4), were used to describe the adsorption isotherms data [19,20].
[please see the revised manuscript]
[please see the revised manuscript]
Where Qm (mg/g) is the maximum adsorption capacity, Ce (mg/L) is the equilibrium concentration of phosphate, KL is the Langmuir constant related to the affinity of phosphate for the binding sites, KF is the Freundlich constant related to the adsorption capacity, and n is a heterogeneity factor related to the heterogeneous surfaces of the absorbent.
2.6 Adsorption Thermodynamics
Tests were performed to investigate the thermodynamics of the inorganic and organic phosphate adsorption by the Fe-Mn plaque powder. In each test, 0.1 g Fe-Mn plaque powder was added to 200 mL solution containing inorganic or organic phosphate with an initial phosphorus concentration of 6 mg/L at pH 7.0 in a 250 mL conical flask. The conical flasks were agitated at 120-130 rpm in a mechanical orbital shaker at 293, 298, 303, 308, and 313 K for 24 h, then each solution was passed through a 0.45 μm polycarbonate membrane filter and the residual inorganic and organic phosphorus concentrations were determined.
The effects of temperature on inorganic and organic phosphate adsorption by the Fe-Mn plaques were investigated by calculating the Gibbs free energy (ΔG0), enthalpy change (ΔH0), and entropy change (ΔS0) using Equations (5) - (7).
[please see the revised manuscript]
[please see the revised manuscript]
[please see the revised manuscript]
Where Kd is the adsorption distribution coefficient calculated from the ratio between the amounts of inorganic or organic phosphate adsorbed (Qe) and the phosphorus concentration (Ce) at equilibrium, R (8.314 J/mol/K) is the ideal gas constant and T (K) is the temperature in Kelvin.
2.7 Competitive Adsorption
Competitive adsorption between inorganic and organic phosphate was investigated by performing a series of tests. In each test, 0.1 g Fe-Mn plaque powder and 200 mL solution containing 6 mg/L inorganic phosphate at pH 7.0 were added to a 250 mL conical flasks. Organic phosphate was added to different flasks to give inorganic to organic phosphate molar ratios of 1:1, 2:1, and 3:1. The flasks were agitated for 24 h at 120~130 rpm using a mechanical orbital shaker at 298 K, then each solution was passed through a 0.45 μm polycarbonate membrane filter and the residual inorganic and organic phosphorus concentrations were determined.
Each test described above was performed in triplicate to minimize errors and the mean inorganic and organic phosphate concentrations were used in the calculations.
Point 3
2.3 The subsection is very general, please detail it.
Response 3: Thanks for the reviewer’s kind suggestion. The 2.3 subsection has been detailed and expanded in the revised manuscript as follows.
The reed roots with and without Fe-Mn plaques attached were examined by SEM using an S-3500N instrument (Hitachi High-Technologies). Before being examined, the samples were sputtering coated with gold and palladium for 45 s using a Quorum Polaron SC7620 mini-sputter coater (Quorum Technologies Ltd, East Sussex, UK) to decrease charging effects inside the microscope [16].
The electron binding energies and the oxidation states of iron and manganese in the plaques were determined by XPS using a Shimadzu ESCA-lab-220i-XL instrument (Shimadzu, Kyoto, Japan) using monochromatized Alkα X-rays at 1486.4 eV.
Freeze dried Fe-Mn plaque samples before and after inorganic and organic phosphate had been adsorbed by plaques were analyzed by FTIR. Each sample was mixed with spectral grade KBr at a weight ratio of 100:1 and pressed to form a disk. The disks were analyzed using a Nicolet 6700 FTIR spectrometer (Thermo Fisher Scientific, Waltham, MA, USA). FTIR spectra over the range 4000 - 500 cm-1 were acquired and the functional groups in the samples were identified.
Point 4
Some parts of methodology seems to be an aims of the study… l. 82-83
Response 4: Thanks for the reviewer’s comment. The parts of methodology which seems to be the aims of study have been moved to in line 59 to 66 in the last paragragh of Introduction chapter in the revised manuscript, as follows.
In this study, Fe-Mn plaques were extracted from reed roots from a long-established wetland, and the abilities of the plaques to adsorb inorganic and organic phosphates were assessed. The surface morphology and structure of the roots were assessed by scanning electron microscopy (SEM), and the electron binding energies and the oxidation states of Fe and Mn in plaques were investigated by X-ray photoelectron spectroscopy (XPS). The functional groups in Fe-Mn plaques were investigated by Fourier-transform infrared (FTIR) spectroscopy. The kinetics, isotherms, and thermodynamics of inorganic and organic phosphate adsorption by Fe-Mn plaques were also assessed.
Point 5
2.4-2.6 How many repetitions you did?
Response 5: Thanks for the reviewer’s professional question. To obtain the appropriate data for analysis and eliminate the possible errors, adsorption kinetics, isotherms and thermodynamis experiments had been repeated 3 times with three parallel samples. And mean concentrations were obtained to analyze for all batch experiments mentioned above. In order to express more clearer, the related description has been added in line 162-163 in the revised manuscript.
Point 6
l. 96 designed time intervals – please specified
Response 6: Thanks for the reviewer’s kind remind. The designed time intervals (5min, 10min, 20min, 30min, 1h, 2h, 4h, 8h, 16h, and 24h) has been added in line 109-110 in the revised manuscript.
Point 7
Results and Discussion
Fig 1. What is the scale of a and b? It seems to be different and maybe that the reason of differences.
Response 7: Thanks for the reviewer’s good question. The scale of (a) and (b) in Fig 1 was 1:2 in the former version. To make the scale consistent, the photo (a) has been amplified and replaced in Fig. 1 in the revised manuscript.
[please see the revised manuscript]
Fig 1 The reed roots (a) with Fe-Mn plaques, (b) without Fe-Mn plaques
Point 8
l.142-143 this is not a part of results and discussion . It more lake a aim… Please rewrite it.
Response 8: Thanks for the reviewer’s nice suggestion. The parts reviewer mentioned as the aim of XPS analysis have been moved to the last paragragh of Introduction chapter in the revised manuscript.
Point 9
l.185-192 This a methodology!
Response 9: Thanks for the reviewer’s kind remind. This part has been moved to Materials and Methods chapter which could be observed in line 117-124 in the revised manuscript.
Point 10
l.205-212 This a methodology!
Response 10: Thanks for the reviewer’s kind remind. This part has been moved to Materials and Methods chapter which could be observed in line 133-136 in the revised manuscript.
Point 11
l.231-241 This a methodology!
Response 11: Thanks for the reviewer’s kind remind. This part has been moved to Materials and Methods chapter which could be observed in line 145-153 in the revised manuscript.
Point 12
Generally, there is lack of discussion of obtained results. Authors only focused on results. Please improve it.
Response 12: Thanks for the reviewer’s good suggestion. The discussion of obtained results have been improved and could be observed in the revised manuscript, for example:
Line 206-209: Fast adsorption of phosphate (i.e., in the first 60 min of our tests) means that the hydraulic retention time (important for continuous operation of waterfront wetland system) will decrease greatly when effluent enter a wetland system.
Line 234-237: The excellent adsorption capacity of the Fe-Mn plaques shows great potential for wetland systems to effectively remove phosphates from polluted water and phosphate removal can be promoted by regulating and controlling Fe-Mn plaques.
Line 260-263: The adsorption characteristics described above indicate that Fe-Mn plaques on plant roots in wetlands play important roles on phosphorus removal at low temperatures. This will offset the limited degrees to which microbial assimilation and adsorption by plants remove phosphorus.
Point 13
Please add the part of adsorption mechanism.
Response 13: Thanks for the reviewer’s nice suggestion. The adsorption mechanism combined with FTIR analysis has been supplemented in line 264 to 286 in the revised manuscript, as follows.
3.5 Adsorption Mechanism
Functional groups on the surfaces of iron and manganese particles commonly participate in the adsorption process and provide abundant adsorption sites for phosphorus. The mechanisms though which inorganic and organic phosphates were adsorbed by the Fe-Mn plaques were investigated by FTIR spectroscopy. The FTIR spectra of KBr pressed-disk containing Fe-Mn plaques before and after inorganic and organic phosphates had been adsorbed were obtained and are shown in Figure 6.
[please see the revised manuscript]
Figure 6. The FTIR spectra of Fe-Mn plaque before and after adsorption.
Bands around 3212 and 1650 cm-1 were assigned to the hydroxyl groups (O-H) stretching vibration and water molecular bending vibration, respectively [33]. A band at 1130 cm-1 was assigned to Fe-OH bending vibration [34,35], and a band at 1438 cm-1 was assigned to hydroxyl groups attached to MnO2 [18]. The Fe-OH bending band (at 1130 cm-1) was weakened and a peak at 1048 cm-1 assigned to P-O vibrations was strengthened when inorganic and organic phosphate become adsorbed, suggesting that surface complexation reactions occurred and that hydroxyl groups attached to the Fe-Mn plaques may have been replaced with H2PO4- or HPO42-, (i.e., phosphate became bound to the Fe-Mn plaques through ligand exchange at pH 7.0) [27,36]. Possible adsorption mechanisms were determined taking the molecular structures of the phosphate species, the changes in the functional groups on the Fe-Mn plaques with and without adsorbed, and the characteristics of the chemisorption process into account, and the mechanisms are shown in Scheme 1.
[please see the revised manuscript]
Scheme 1. Schematic diagram of possible phosphate adsorption reaction.
Point 14
What is the utilization part of yours study?
Response 14: Thanks for the reviewer’s question. In China, phosphorus residues in wastewater treatment plants effluent are generally removed primarily using a wetland system before the effluent is discharged into a water body. In such wetland systems, phosphorus is removed by plant uptake, media adsorption and microorganism assimilation. Media adsorption is usually limited because much of the solid matter is inert and has a poor adsorption capacity. Assimilation by microorganisms contributes little to the removal of total phosphorus because the phosphorus will be released back to the water once the microorganisms die. Plant uptake is normally considered to affect phosphorus removal little until the plants are harvested. However, oxygen can be released from the dense root networks of plants in wetlands, creating an oxidizing rhizosphere environment. This can cause Fe2+ and Mn2+ in the rhizosphere soil or media to become oxidized to Fe3+ and Mn4+, respectively. The Fe3+ and Mn4+ can precipitate and accumulate on the root surfaces, to form features called iron manganese (Fe-Mn) plaques. Fe-Mn plaques on hydrophyte root surfaces can immobilize various nutrients and metalloids. Wang et al. found Arundo donax Linn and Typha latifolia roots had higher phosphorus contents when iron plaques were presented than when iron plaques were not present. Chong et al. found that amorphous iron oxide on root surfaces caused phosphorus to accumulate in the rhizosphere.
The study is a fundamental research of discovering the effect of Fe-Mn plaque on phosphorus removal in wetland systems. The contribution of inorganic and organic phosphates removal by Fe-Mn plaque was specifically figured out in this paper, which can provide guidance for the application of the long-term effective and sustainable phosphorus removal through controlling the interior habitat to promote the formation of Fe-Mn plaque in waterfront wetland systems. Also, it has been found that Fe-Mn plaque on plant roots in wetland plays a considerable role on phosphorus removal under low-temperature conditions, offsetting the limited phosphorus removal by microbial assimilation and plant adsorption. Then it is a direction to regulating and controlling Fe-Mn plaque generation by optimizing the operation for a sustainable phosphorus removal in wetland systems.
The aim of the study for utilization has been added in line 55-58 and some discussion for utilization of the study has been added in line 234-237 and 260-263 in the revised manuscript.
Point 15
l.174-175 this is a repetition.
Response 15: Thanks for the reviewer’s kind remind. The repetition part has been deleted in the revised manuscript.
Point 16
Table 3. FMO – what is this abbreviation?
Response 16: Thanks for the reviewer’s question. The FMO is the abbreviation of Fe-Mn oxide, which has been replaced by the full name in the revised manuscript.
Point 17
Table 4 instead Table 3 (is doubled)
Response 17: We are so sorry for the careless mistake. The name of Table 4 has been corrected in the revised manuscript.
Point 18
l. 249-251 –repetition, that data is presented in Table 4. Please change it
Response 18: Thanks for the reviewer’s kind remind. The repeated data has been deleted in the revised manuscript.
Point 19
Conclusion
There were no mention of chemisorption and monolayer adsorption in the Results and Discussion part…
Response 19: Thanks for the reivewer’s comment. The related content of chemisorption and monolayer adsorption can be observed in the revised manuscript as follows:
Line 210-216: The kinetic parameters for the pseudo-first-order and pseudo-second-order models are presented in Table 1. As shown in Table 1, the experimental data were better described by the pseudo-second-order model than the pseudo-first-order model. The R2 values for inorganic and organic phosphates were 0.989 and 0.965, respectively. This indicated that both inorganic and organic phosphates may be adsorbed by Fe-Mn plaque through chemisorption involving covalent forces by sharing or exchanging of electrons between the Fe-Mn plaques and phosphate, as described by [26,27].
Line 225-227: The Langmuir model generally describes monolayer adsorption, so the results suggest that the Fe-Mn plaques the active sites distributed homogeneously and that the adsorption energies for different sites were similar and no other interactions occurred [19].
Point 20
Generally, the manuscript is very chaotic and have to be strictly rewritten.
Response 20: Thanks for the reviewer’s comment. The entire manuscript has been rewritten sharply and the structure has been adjusted. The language has been revised by a native English speaker to enhance the readability of the paper.
Author Response File: 
Author Response.docx
Reviewer 2 Report
Adsorption behavior, such as adsorption kinetics, adsorption isotherms,
adsorption thermodynamics, competitive adsorption, of phosphorus by
Iron-manganese plaque on reed roots was clearly stated. However, the originality
of this approach and new findings were not clearly stated. The appropriate
explanation should be added in "Introduction".
Author Response
Response to Reviewer 2 Comments
Point 1
Adsorption behavior, such as adsorption kinetics, adsorption isotherms, adsorption thermodynamics, competitive adsorption, of phosphorus by Iron-manganese plaque on reed roots was clearly stated. However, the originality of this approach and new findings were not clearly stated. The appropriate explanation should be added in "Introduction".
Response 1: Thanks for the reviewer’s nice question. As we know, Fe-Mn plaques on hydrophyte root surfaces can immobilize various nutrients and metalloids. Wang et al. found Arundo donax Linn and Typha latifolia roots had higher phosphorus contents when iron plaques were presented than when iron plaques were not present. Chong et al. found that amorphous iron oxide on root surfaces caused phosphorus to accumulate in the rhizosphere. However, the phosphorus adsorption behaviors and mechanisms by Fe-Mn plaques are not clearly stated, and the influence between inorganic and organic phosphate is ignored. Therefore, the contributions of Fe-Mn plaques to the removal of inorganic and organic phosphates in wetland systems need to be assessed to improve our understanding of how wetland system habitats may be managed to promote Fe-Mn plaques formation to effectively and sustainably remove phosphorus from water. The appropriate explanation has been added in the Introduction in line 53-58.
Author Response File: Author Response.docx
Reviewer 3 Report
The manuscript by Zhu et al describes the results of a study purported to investigate the effect of Fe-Mn plaque on Phragmites roots in removing inorganic and organic forms of P. Such information is important to know for predicting removal abilities and in controlling wastewater pollution with high P loads. While the paper does provide some of this information, it could provide more. In addition, there are numerous mistakes that need to be corrected prior to the manuscript being publishable.
First, the English, while not terrible, does need some improvement. There are numerous examples of wordy, awkward writing. For example, the entire last paragraph of the Introduction is too wordy and should be written in a more concise manner. I strongly recommend that the authors have a native English speaker edit the revised manuscript before re-submission.
Second, there is information that belongs in the Methods that is actually in the Results/Discussion. Specifically, the information dealing with adsorption kinetics (lines 185-192), adsorption isotherms (lines 205-212) and adsorption thermodynamics (lines 231-241) describe how data were analyzed. Again, such information should be in the Methods. Also, there was no mention of analyzing adsorption thermodynamics in the Methods; this is interesting and important information but it should be mentioned in the Methods.
Third, the paper as is seems rather detail-oriented with no information whether the Fe-Mn plaques significantly remove P compared to plants without the plaque. There is a mention about this concerning the FTIR results but this is only a qualitative, descriptive mention with no analyses behind it. Having such a comparison would greatly strengthen the paper. I would strongly recommend that, if it is possible, the authors quantify the FTIR peaks for the plants with and without the plaques and then analyze these data statistically to see whether the differences are statistically significant or not.. It is too late for this paper for the authors to run other experiments making these comparisons (if the authors have such data then they may wish to include them) but would be an important next step to do.
Fourth, the Conclusions is actually only a summary of the results. A conclusion section should not only repeat the important results but go further and expand the focus of the section to what would be the next steps to study as well as how or why these results are important. A little speculation would not be a bad thing.
Author Response
Response to Reviewer 3 Comments
Point 1:
The English, while not terrible, does need some improvement. There are numerous examples of wordy, awkward writing. For example, the entire last paragraph of the Introduction is too wordy and should be written in a more concise manner. I strongly recommend that the authors have a native English speaker edit the revised manuscript before re-submission.
Response 1: Thanks for the reviewer’s nice suggestion. The language of the manuscript has been revised by a native English speaker to enhance the readability of the paper. And the last paragraph has been rewritten in the revised manuscript.
Line 59-66: In this study, Fe-Mn plaques were extracted from reed roots from a long-established wetland, and the abilities of the plaques to adsorb inorganic and organic phosphates were assessed. The surface morphology and structure of the roots were assessed by scanning electron microscopy (SEM), and the electron binding energies and the oxidation states of Fe and Mn in plaques were investigated by X-ray photoelectron spectroscopy (XPS). The functional groups in Fe-Mn plaques were investigated by Fourier-transform infrared (FTIR) spectroscopy. The kinetics, isotherms, and thermodynamics of inorganic and organic phosphate adsorption by Fe-Mn plaques were also assessed.
Point 2:
There is information that belongs in the Methods that is actually in the Results/Discussion. Specifically, the information dealing with adsorption kinetics (lines 185-192), adsorption isotherms (lines 205-212) and adsorption thermodynamics (lines 231-241) describe how data were analyzed. Again, such information should be in the Methods. Also, there was no mention of analyzing adsorption thermodynamics in the Methods; this is interesting and important information but it should be mentioned in the Methods.
Response 2: Thanks for the reviewer’s constructive comment. The Materials and Methods chapter has been strictly rewritten. The information that belongs in the Methods has been moved from the Results/Discussion to Materials and Methods chapter. And the analysis of adsorption thermodynamics was supplemented in the Materials and Methods chapter in the revised manuscript, as follows:
2. Materials and Methods
2.1 Chemicals
All chemicals were of analytical reagent grade and were purchased from Beijing Chemical Co. (Beijing, China). Solutions of inorganic and organic phosphates for use in the adsorption tests were prepared by dissolving potassium dihydrogen phosphate (KH2PO4) and adenosine-5’-monophosphate (C10H14N5O7P), respectively, in ultrapure water. The ultrapure water (18.2 ΩM·cm-1) used throughout the study was prepared using a Millipore system (Merck, Darmstadt, Germany).
2.2 Fe-Mn Plaque Collection
The rReeds with roots were collected from a wetland at the Beijing University of Civil Engineering and Architecture (Beijing, China). The wetland is used to treat effluent from a sewage treatment plant. The roots were cut from the reeds and washed thoroughly with ultrapure water. The roots were then placed in a beaker containing ultrapure water and ultrasonicated for 5h using a KQ-500B instrument (Kun Shan Ultrasonic Instruments Co., Ltd, Kun Shan, China). This caused the Fe-Mn plaques to separate from the root surfaces and become suspended in the water. The process was repeated until sufficient suspension was obtained to perform the planned tests. The suspended plaques were reddish brown. The suspension was evaporated and freeze dried to give dry Fe-Mn powder, which was stored in a desiccator. The iron and manganese contents of the Fe-Mn plaque powder were determined by acid digesting and then analyzing the solution using a Hitachi Z-2010 atomic absorption spectrometer (Hitachi High-Technologies, Tokyo, Japan). The total phosphorus and inorganic phosphorus contents of the Fe-Mn plaque powder were determined and used as the background concentrations (i.e., before adsorption experiments were performed). The weights of the roots were determined before the ultrasonic extraction process was performed.
2.3 Characterization
The reed roots with and without Fe-Mn plaques attached were examined by SEM using an S-3500N instrument (Hitachi High-Technologies). Before being examined, the samples were sputtering coated with gold and palladium for 45 s using a Quorum Polaron SC7620 mini-sputter coater (Quorum Technologies Ltd, East Sussex, UK) to decrease charging effects inside the microscope [16].
The electron binding energies and the oxidation states of iron and manganese in the plaques were determined by XPS using a Shimadzu ESCA-lab-220i-XL instrument (Shimadzu, Kyoto, Japan) using monochromatized Alkα X-rays at 1486.4 eV.
Freeze dried Fe-Mn plaque samples before and after inorganic and organic phosphate had been adsorbed by plaques were analyzed by FTIR. Each sample was mixed with spectral grade KBr at a weight ratio of 100:1 and pressed to form a disk. The disks were analyzed using a Nicolet 6700 FTIR spectrometer (Thermo Fisher Scientific, Waltham, MA, USA). FTIR spectra over the range 4000 - 500 cm-1 were acquired and the functional groups in the samples were identified.
2.4 Adsorption Kinetics
kinetics of inorganic and organic phosphate adsorption by the Fe-Mn plaque powder. 0.1 g Fe-Mn plaque powder was added to 200 mL solutions containing 6 mg/L inorganic or organic phosphate at pH 7.0 in a 250 ml conical flask. The conical flasks were agitated at 120-130 rpm using a mechanical orbital shaker at 298 K. An aliquot of the solution in each test was sampled at each specified time intervals (5min, 10min, 20min, 30min, 1h, 2h, 4h, 8h, 16h and 24h) and passed through a 0.45 μm polycarbonate membrane filter, then the inorganic phosphorus concentration was determined using an ammonium molybdate spectrophotometric method and the total phosphorus concentration was determined using an alkaline potassium persulfate digestion spectrophotometric method, using a Sunny Hengping Instrument 752 ultraviolet visible spectrophotometer (Sunny Hengping Instrument, Shanghai, China) at wavelength of 700 nm. The organic phosphorus concentration was defined as the difference between the total phosphorus and inorganic phosphorus.
The mechanisms and steps controlling adsorption were analyzed by fitting the pseudo-first-order and pseudo-second-order models expressed in the Equations (1) and (2) [17,18], respectively, to the kinetics data.
[Please see the revised manuscript]
[Please see the revised manuscript]
Where t (min) is the contact time, Qe (mg/g) and Qt (mg/g) are the amounts of phosphate adsorbed at equilibrium time and time t, and K1 (1/min) and K2 (g/mg/min) are the pseudo-first-order and pseudo-second-order adsorption rate constants, respectively.
2.5 Adsorption Isotherms
Tests were performed to investigate the isotherms for the inorganic and organic phosphate adsorption by the Fe-Mn plaque powder. In each test, 0.1 g Fe-Mn plaque powder was added to 200 mL solution containing inorganic or organic phosphate in a 250 mL conical flask. Tests were performed using initial phosphorus concentrations between 0.1 mg/L and 6 mg/L at pH 7.0. The conical flasks were agitated at 120-130 rpm using a mechanical orbital shaker at 298 K. After 24 h, each solution was passed through a 0.45 μm polycarbonate membrane filter and the residual inorganic and organic phosphorus concentrations were determined.
The Langmuir and Freundlich models, shown in Equations (3) and (4), were used to describe the adsorption isotherms data [19,20].
[Please see the revised manuscript]
[Please see the revised manuscript]
Where Qm (mg/g) is the maximum adsorption capacity, Ce (mg/L) is the equilibrium concentration of phosphate, KL is the Langmuir constant related to the affinity of phosphate for the binding sites, KF is the Freundlich constant related to the adsorption capacity, and n is a heterogeneity factor related to the heterogeneous surfaces of the absorbent.
2.6 Adsorption Thermodynamics
Tests were performed to investigate the thermodynamics of the inorganic and organic phosphate adsorption by the Fe-Mn plaque powder. In each test, 0.1 g Fe-Mn plaque powder was added to 200 mL solution containing inorganic or organic phosphate with an initial phosphorus concentration of 6 mg/L at pH 7.0 in a 250 mL conical flask. The conical flasks were agitated at 120-130 rpm in a mechanical orbital shaker at 293, 298, 303, 308, and 313 K for 24 h, then each solution was passed through a 0.45 μm polycarbonate membrane filter and the residual inorganic and organic phosphorus concentrations were determined.
The effects of temperature on inorganic and organic phosphate adsorption by the Fe-Mn plaques were investigated by calculating the Gibbs free energy (ΔG0), enthalpy change (ΔH0), and entropy change (ΔS0) using Equations (5) - (7).
[Please see the revised manuscript]
[Please see the revised manuscript]
[Please see the revised manuscript]
Where Kd is the adsorption distribution coefficient calculated from the ratio between the amounts of inorganic or organic phosphate adsorbed (Qe) and the phosphorus concentration (Ce) at equilibrium, R (8.314 J/mol/K) is the ideal gas constant and T (K) is the temperature in Kelvin.
2.7 Competitive Adsorption
Competitive adsorption between inorganic and organic phosphate was investigated by performing a series of tests. In each test, 0.1 g Fe-Mn plaque powder and 200 mL solution containing 6 mg/L inorganic phosphate at pH 7.0 were added to a 250 mL conical flasks. Organic phosphate was added to different flasks to give inorganic to organic phosphate molar ratios of 1:1, 2:1, and 3:1. The flasks were agitated for 24 h at 120~130 rpm using a mechanical orbital shaker at 298 K, then each solution was passed through a 0.45 μm polycarbonate membrane filter and the residual inorganic and organic phosphorus concentrations were determined.
Each test described above was performed in triplicate to minimize errors and the mean inorganic and organic phosphate concentrations were used in the calculations.
Point 3
The paper as is seems rather detail-oriented with no information whether the Fe-Mn plaques significantly remove P compared to plants without the plaque. There is a mention about this concerning the FTIR results but this is only a qualitative, descriptive mention with no analyses behind it. Having such a comparison would greatly strengthen the paper. I would strongly recommend that, if it is possible, the authors quantify the FTIR peaks for the plants with and without the plaques and then analyze these data statistically to see whether the differences are statistically significant or not.. It is too late for this paper for the authors to run other experiments making these comparisons (if the authors have such data then they may wish to include them) but would be an important next step to do.
Response 3: Thanks for the reviewer’s professional and valuable suggestion. The comparison of FTIR peaks for the plants with and without Fe-Mn plaque can provide the significant differences statistically for deep discussion. Unfortunately, the point we focused on before was the comparison of FTIR analysis between inorganic and organic phospshate removal by Fe-Mn plaque to illustrate the mechanism of phosphorus removal. Then the FTIR analysis of roots with and without Fe-Mn plaque was ignored. Possibly, it is too late for this paper to run other experiments making these comparisons. We think that will be understood by the reviewer. We will compare that in the next step. And we are grateful for the reviewer’s kind remind.
Point 4
The Conclusions is actually only a summary of the results. A conclusion section should not only repeat the important results but go further and expand the focus of the section to what would be the next steps to study as well as how or why these results are important. A little speculation would not be a bad thing.
Response 4: Thanks for the reviewer’s professional suggestion. The Conclusion chapther has been modified and some direction of next step has been added in, which is shown in the revised manuscript as follows.
4. Conclusions
Fe-Mn plaques were extracted from reed roots. The plaques were characterized and then batch tests were performed to investigate inorganic and organic phosphate adsorption from aqueous solutions by the plaques. SEM images indicated that the roots with Fe-Mn plaques attached were rough and had heterogeneous porous structures and fine particles attached. XPS indicated that the iron and manganese in the Fe-Mn plaques were predominantly in the forms Fe(III) and Mn(IV), respectively. FTIR spectroscopy revealed that the adsorption process of phosphate was caused by hydroxyl substitution and inner-sphere surface complexion of orthophosphate or organophosphate ion groups. Pseudo-second order model described both inorganic and organic phosphate adsorption kinetic well, indicating that both inorganic and organic phosphate adsorption processes were chemisorption. The Langmuir isotherm model fitted the adsorption data well, indicating that monolayer adsorption occurred. And the maximum inorganic and organic phosphate adsorption capacities at 298 K were 7.69 and 3.66 mg/g , respectively. Thermodynamic data indicated that both inorganic and organic phosphate adsorption processes were spontaneous and exothermic. The innovative use of low-cost, natural, and the efficient adsorbents offers great potential for the sustainable removal of phosphorus from wastewater. Phosphorus removal by Fe-Mn plaques on plant roots should be enhanced by managing wetland habitats to promote Fe-Mn plaques production.
Author Response File: Author Response.docx
Reviewer 4 Report
Title: Adsorption Behavior of Inorganic and Organic Phosphate by Iron-manganese Plaque on Reed Roots in Wetland
This manuscript provide an report on the adsorption behavior of phosphate by Iron-manganese plaque on plant roots in wetland. The topic is interesting and important. The authors sampled and extracted the iron and manganese plaque (Fe-Mn plaque) from the reed roots in a wetland, and several methods (SEM, XPS, FTIR) were conducted to characterize it. The adsorption kinetic, isotherms, thermodynamics and competitive adsorption of inorganic/organic phosphate were investigated to illustrate the adsorption behaviors. In general, the work is novel and all required experiment are well done, the presentation of results and discussion are well organized. It seems to be suitable for publication in Sustainability. However, they are few points need to be clarified before it can be accept.
- The method of DCB is usually to extract the Fe-Mn plaque from roots, why the authors selected the physical method of an ultrasonic cleaner?
- Please explain the reason for maximum initial concentration (6 mg/L) of phosphorus.
Author Response
Response to Reviewer 4 Comments
Point 1
The method of DCB is usually to extract the Fe-Mn plaque from roots, why the authors selected the physical method of an ultrasonic cleaner?
Response 1: The reviewer posed a professional question. DCB (dithionite-citrate-bicarbonate) method was proposed by Gregory J. Taylor and A. A. Crowder in 1983, which is a chemical extract method widly used by the following researchers. However, it intended to investigate the adsorption behaviours of phosphorus by Fe-Mn plaque in our research, which could possibly be effected by the chemicals employed in the DCB method. Then a physical method like ultrasonic cleaner extraction for avoiding the unpredictable affect on adsorption behaviours was used in the study.
Ref: Taylor, G. J.; Crowder, A. A. Use of the DCB Technique for Extraction of Hydrous Iron Oxides from Roots of Wetland Plants. Am. J. Bot. 1983, 70, 1254, doi:10.2307/2443295.
Point 2
Please explain the reason for maximum initial concentration (6 mg/L) of phosphorus.
Response 2: The reviewer posed a professional question. The initial concentration of phosphorus depended on the concentration range for phosphorus in the sewage in China, which varies from 2 to 8 mg/L. Then we choose 6 mg/L as the maximum value in the manuscript.
Author Response File: Author Response.docx
Round 2
Reviewer 1 Report
Compared to first version, the quality is improved. The authors have revised the questions according to my comments, so it can be accepted in this form.
Author Response
Response to reviewer 1:
Point 1: Compared to first version, the quality is improved. The authors have revised the questions according to my comments, so it can be accepted in this form.
Response: We are glad to have reviewer’s approval, thanks again for reviewer’s valuable suggestions.
Reviewer 3 Report
This is a much better version of the manuscript. I thank the authors for making the recommended changes. The only thing of note would be a few changes in the language, in particular:
line 178: should read - 1(c) and 1(d), the root surfaces with and without Fe-Mn plagues were distinctly different.
line 208: last word of the line should be Larger
Author Response
Response to reviewer 3:
Point 1: This is a much better version of the manuscript. I thank the authors for making the recommended changes. The only thing of note would be a few changes in the language, in particular:
line 178: should read - 1(c) and 1(d), the root surfaces with and without Fe-Mn plagues were distinctly different.
line 208: last word of the line should be Larger
Response: Thanks for reviewer’s recognition and suggestion. A few changes in language have been made in the revised manuscript, in particular:
Line 178: As shown in Figure 1(c) and 1(d), that the root surfaces with and without Fe-Mn plaques were distinctly different.
Line 208: Larger amounts of inorganic phosphate than organic phosphate were adsorbed, indicating that inorganic phosphate was more easily absorbed than organic phosphate by the Fe-Mn plaques.
