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Peer-Review Record

A High Manganese-Tolerant Pseudomonas sp. Strain Isolated from Metallurgical Waste Heap Can Be a Tool for Enhancing Manganese Removal from Contaminated Soil

Appl. Sci. 2020, 10(16), 5717; https://doi.org/10.3390/app10165717
Reviewer 1: Adarsh Kumar
Reviewer 2: Kehinde Erinle
Appl. Sci. 2020, 10(16), 5717; https://doi.org/10.3390/app10165717
Received: 24 July 2020 / Revised: 10 August 2020 / Accepted: 13 August 2020 / Published: 18 August 2020

Round 1

Reviewer 1 Report

The article titled "A High Manganese-Tolerance Pseudomonas sp. Strain Isolated From Metallurgical Waste Heap Can Be A Tool For Enhancing Manganese Removal From Contaminated Soil" is focused on how a Mn accumulating bacteria can help in removal of Mn from the Mn contaminated site. The work is presented well and fairly written. Before its acceptance I suggest some comments which need to be addressed in revised MS:

L90-91 Please add a recent reference .

L201 What does d.w. mean here? Please specify and if it is dry weight write in capital letters.

Table 1 It is better to write word "Strain" before any numbers.

L238 Please specify the country name.

L251 Please write the alphabets a , b, c in a sequence and delete the repeated punctuation.

Conclusion needs to be re-written by addressing the main findings.

Cheers

Author Response

Dear Reviewer,

Thank you for all of your detailed comments, suggestions, and editing corrections. They helped us to improve our manuscript (applsci-893758). All missing information, suggestions, and references were added to the manuscript and are indicated in red.

Comment 1: L90-91 Please add a recent reference. The references were added (line 95 in the revised manuscript).

Comment 2: L201 What does d.w. mean here? Please specify and if it is dry weight write in capital letters. The abbreviation dry weigh was revised into D.W. (line 210 in the revised manuscript).

Comment 3: Table 1 It is better to write word "Strain" before any numbers. The word “strain” was used in the title row of the first column, therefore its using before any numbers in the table is unnecessary (Table 2 in the revised manuscript).

Comment 4: L238 Please specify the country name. The name previously specified in lines 236-237 (line 248 in the revised manuscript).

Comment 5: L251 Please write the alphabets a , b, c in a sequence and delete the repeated punctuation. The Figure 2 description was revised according to the Reviewer’s suggestion  (257-258 in the revised manuscript).

Comment 6: Conclusion needs to be re-written by addressing the main findings. The conclusions were revised according to the Reviewer’s suggestion (412-423 in the revised manuscript).

Reviewer 2 Report

The paper titled “A High Manganese-Tolerance Pseudomonas sp. Strain Isolated From Metallurgical Waste Heap Can Be A Tool For Enhancing Manganese Removal From Contaminated Soil” evaluates the efficiency of a microbial strain 2De in bioremediating Mn contamination in contaminated media (soil slurry and liquid). Effect of the Mn adsorption on the microbial properties such as protein was determined. The work is well described and experiments well conducted with clarity. The findings in the papers are also interesting. However, the authors need to succinctly polish to English language. The paper is worth publishing after significant revision.

A very important question is the feasibility of applying the microbe in soil medium rather than in a slurry of 1 to 6% soil (w/v)? how would the result be different thus? In the light of this, the authors should not generalise the results as a tool to “remove and recover Mn from contaminated soil” as the experiment only contained 6% soil in a solution as opposed to a soil matrix. It appears that the experiment carried out in section 2.7 was not presented in the paper, or not entirely presented? In section 2.8, why did authors choose to use 4% but not 6% soil? Authors need to revise the figures again. For example, figures 3 and 4 do not have descriptors, though the reader can assume the columns are 1, 2, 4 and 6%, and figure 5 seems to have an incorrect descriptor. Other comments from each section are listed below.

Abstract

Line 14-16. The sentence should be rewritten as "However, its extensive applications have generated a great amount of manganese waste, which has become an ecological problem and has led to a decrease in natural resources."

line 21: "its ability for Mn sorption"

Introduction

line 43: "some of these wastes are"

line 54: "gaining more interest"

line 58: do you mean both methods are used by microbes or either of both?

line 69: "for treatment of metallurgical and mining wastewater"

line 77: "The main aim of this work was to investigate the removal potential ..."

Materials and methods

line 87: "soil samples ... were" or "soil sample ... was"

line 90: "in the soil sample was"

line 91-92: include volume to mass ratio of extractant to soil used

lines 98, 104, and many other places: note superscripts

line 106: each set of what? the microbes?

line 122-123: "first and last day of the experiment" means "before and after the 14 days incubation"?

line 151: it would be good for the reader to be reminded that the soil used here was previously sterilized to eliminate other microbial presence.

line 152: did you note the initial Mn concentration in the soil?

line 174: medium K was used here as a blank (soiless) control?

Results and discussion

line 202: would it not be good to show (say in a table) the concentrations of these metals?

line 245-247: I feel this sentence should rather be in the materials and method section?

line 283-285: this sentence should be in the materials and methods, and not repeated here.

Fig. 3: please show the keys. which column represents what?

Fig. 4: why are the alphabets missing on the columns?

line 318: please delete "what"

line 325-327: please provide reference(s)

line 335-336: does the bacteria only produce biosurfactant at 6% soil, but not at 2% and 4%? could you explain why the result was different only for 6%. If the phenomenon is linked with high Mn extraction efficiency (line 337), then I would suppose the surface tension (Fig 4b) should increase with increasing Mn concentration before reaching 6%.

line 359-361: this is only a repetition of the materials and methods. Should be deleted. I understand it is to recall the reader to the focus of the section, but since there is already a subtitle, this is just sufficient.

Figure 5: looks like this figure is not complete or needs to adjust the keys? why did you present only 4% soil, how about 6% that produced highest biosurfactant in the microbes as opined in previous paragraph?

line 371: "store"? since you stated in line 367 that the Mn was "cell-surface bound", would you call this storage? I would think storage refers to imbibition of the Mn? Please explain.

line 378-382: does this suggest that the rate of Mn attachment/biosorption, which took 3 days to reach about 55% (as in Fig 6), is faster than the rate of desorption that took 15 days to reach about 9%? does the medium (solution without soil vs with soil) also contribute to the rate of Mn attachment on the microbe?

Figure 6: why did you present only 4% soil, how about 6% that produced highest biosurfactant in the microbes?

Conclusion

I suggest the opening sentence be delete since you did not work on different soils and the effects of soil conditions on the microbe(s).

line 400: "applied" not "developed"

line 403: delete "simultaneous"

Author Response

Dear Reviewer,

Thank you for all of your detailed comments, suggestions, and editing corrections. They helped us to improve our manuscript (applsci-893758). All missing information, suggestions, and references were added to the manuscript and are indicated in green.

 

A very important question is the feasibility of applying the microbe in soil medium rather than in a slurry of 1 to 6% soil (w/v)? how would the result be different thus? In the light of this, the authors should not generalise the results as a tool to “remove and recover Mn from contaminated soil” as the experiment only contained 6% soil in a solution as opposed to a soil matrix. To not generalize the results, the sentence was changed into “This study demonstrated that autochthonous 2De strain can be an effective tool to remove and recover Mn from contaminated soil” (lines 27-29 in the revised manuscript).

It appears that the experiment carried out in section 2.7 was not presented in the paper, or not entirely presented? The results of experiment 2.7 were presented in the Results and Discussion in section 2.4 “Removal efficiency”.

In section 2.8, why did authors choose to use 4% but not 6% soil? The answer to this question is given below and highlighted.

Authors need to revise the figures again. For example, figures 3 and 4 do not have descriptors, though the reader can assume the columns are 1, 2, 4 and 6%, and figure 5 seems to have an incorrect descriptor. Other comments from each section are listed below. Thank you for this suggestion. All legends have now been given correctly.

Abstract

Comment 1: Line 14-16. The sentence should be rewritten as "However, its extensive applications have generated a great amount of manganese waste, which has become an ecological problem and has led to a decrease in natural resources." The sentence was revised according to the Reviewer’s suggestion (lines 14-16 in the revised manuscript).

Comment 2: line 21: "its ability for Mn sorption". The sentence was revised according to the Reviewer’s suggestion (line 21 in the revised manuscript).

Introduction

Comment 3: line 43: "some of these wastes are" We can not agree with this suggestion because this sentence was corrected by Elsevier Language Editing Services.

Comment 4: line 54: "gaining more interest". The sentence was revised according to the Reviewer’s suggestion (line 55 in the revised manuscript).

Comment 5: line 58: do you mean both methods are used by microbes or either of both? Both methods are used by microbes. Both living and non-living cells have biosorption. Bioaccumulation happens only in living cells. Living cell removes metals by biosorption and bioaccumulation, while non-living cell removes metals only by biosorption.

Comment 6: line 69: "for treatment of metallurgical and mining wastewater". The sentence was revised according to the Reviewer’s suggestion (lines 69-70 in the revised manuscript).

Comment 7: line 77: "The main aim of this work was to investigate the removal potential ..." The sentence was revised according to the Reviewer’s suggestion (line 78 in the revised manuscript).

Materials and methods

Comment 7: line 87: "soil samples ... were" or "soil sample ... was" The sentence was revised according to the Reviewer’s suggestion (line 88 in the revised manuscript).

Comment 8: line 90: "in the soil sample was" The sentence was revised according to the Reviewer’s suggestion (line 91 in the revised manuscript).

Comment 9: line 91-92: include volume to mass ratio of extractant to soil used. This information was included in the revised manuscript (lines 91-96 in the revised manuscript).

To determine the total manganese content, we used the wet method of sample decomposition with microwave assistance. We tested several variants of liquid oxidizing mixtures based on the available literature (Kalembkiewicz et al., 2008; Kluczka et al., 2012; Smoliński et al., 2009; Volkov et al., 2015). Finally, we chose the most favorable system: 10 cm3 HNO3 (65% m/m) and 5 cm3 HCl (36% m/m) in ratio to 0.5 g of homogenized and dry soil sample.

Kalembkiewicz, J., Sitarz-Palczak, E., Zapała, L., 2008. A study of the chemical forms or species of manganese found in coal fly ash and soil. Microchem. J. 90, 37–43. https://doi.org/10.1016/j.microc.2008.03.003

Kluczka, J., ZoŁotajkin, M., Ciba, J., 2012. Speciation of aluminium in the water and bottom sediment of fish-breeding ponds. Arch. Environ. Prot. 38, 83–96. https://doi.org/10.2478/v10265-012-0007-7

Smoliński, A., Zołotajkin, M., Ciba, J., Dydo, P., Kluczka, J., 2009. PLS-EP algorithm to predict aluminum content in soils of Beskid Mountains region. Chemosphere 76, 565–571. https://doi.org/10.1016/j.chemosphere.2009.02.057

Volkov, A.I., Ossipov, K.B., Seregin, A.N., Zhdanov, P.A., Seregina, I.F., Bolshov, M.A., 2015. Determination of degree of oxidation and forms of manganese compounds in the Ulu-Telyak oxidized ore. Inorg. Mater. 51, 1394–1403. https://doi.org/10.1134/S0020168515140125

Comment 10: lines 98, 104, and many other places: note superscripts. All superscripts were revised according to the Reviewer’s suggestion.

Comment 11: line 106: each set of what? the microbes? The sentence was revised into “Each set containing soil and a specific strain was prepared in triplicate” (lines 110-111 in the revised manuscript).

Comment 12: line 122-123: "first and last day of the experiment" means "before and after the 14 days incubation"? The sentence was revised into: “The analyses were performed before and after the 14 days incubation period” (lines 126-127 in the revised manuscript).

Comment 13: line 151: it would be good for the reader to be reminded that the soil used here was previously sterilized to eliminate other microbial presence. The word “sterile” was added (line 155 in the revised manuscript).

Comment 14: line 152: did you note the initial Mn concentration in the soil? Yes, this information is written inline 210.

Comment 15: line 174: medium K was used here as a blank (soilless) control? Yes.

Results and discussion

Comment 16: line 202: would it not be good to show (say in a table) the concentrations of these metals? According to the Reviewer’s suggestion, a new Table 1 was added (lines 212-213 in the revised manuscript).

Comment 17: line 245-247: I feel this sentence should rather be in the materials and method section? This sentence was moved to Materials and methods (lines 169-173).

Comment 18: line 283-285: this sentence should be in the materials and methods, and not repeated here. This sentence was deleted (lines 288-290 in the revised manuscript).

Comment 19: Fig. 3: please show the keys. which column represents what? We apologize for this mistake. The legend was added.

Comment 20: Fig. 4: why are the alphabets missing on the columns? We apologize for this mistake. The legend was added.

Comment 21: line 318: please delete "what". This word was deleted (line 324 in the revised manuscript).

Comment 22: line 325-327: please provide reference(s). Reference was provided inline 334 (in the revised manuscript; [48]).

Comment 23: line 335-336: does the bacteria only produce biosurfactant at 6% soil, but not at 2% and 4%? could you explain why the result was different only for 6%. The biosurfactant production by bacteria in metal-contaminated soil strongly depends on the chemical composition of the soil. Probably organic and inorganic compounds (including the presence of manganese) which were present in the soil stimulate bacteria to biosurfactant production, however soil at a concentration of 2% and 4% contain insufficient of these compounds, therefore we not observed decrease in surface tension.

If the phenomenon is linked with high Mn extraction efficiency (line 337), then I would suppose the surface tension (Fig 4b) should increase with increasing Mn concentration before reaching 6%. Most of the biosurfactants are able to bind the metal ions and the percentage removal of these metals varied with the different concentration of metals and biosurfactants. Increasing Mn concentration caused decreasing in surface tension resulting from increased biosurfactant production what is in agreement with other studies tested other metals.

Comment 24: line 359-361: this is only a repetition of the materials and methods. Should be deleted. I understand it is to recall the reader to the focus of the section, but since there is already a subtitle, this is just sufficient. This sentence was deleted (lines 365-367 in the revised manuscript).

Comment 25: Figure 5: looks like this figure is not complete or needs to adjust the keys? why did you present only 4% soil, how about 6% that produced highest biosurfactant in the microbes as opined in previous paragraph? The wrong key in Figure 5 was our mistake which occurs during adaptation of the manuscript to the MDPI template, we sorry for this neglect. In the presented study we used soil only at concentrations 4% and not 6% because the aim of this study was to test the hypothesis whether the strain is capable of manganese accumulation and whether the form in which manganese occurs affects the effectiveness of its accumulation by the tested strain. If the soil was at concentration 6%, the results would be similar showing that the strain is able to Mn accumulation.

Comment 26: line 371: "store"? since you stated in line 367 that the Mn was "cell-surface bound", would you call this storage? I would think storage refers to imbibition of the Mn? Please explain. Thank you for this right suggestion, the word “store” was changed into “accumulate” (line 277 in the revised manuscript).

Comment 27: line 378-382: does this suggest that the rate of Mn attachment/biosorption, which took 3 days to reach about 55% (as in Fig 6), is faster than the rate of desorption that took 15 days to reach about 9%? Yes and this was explained in lines 385-388. Does the medium (solution without soil vs with soil) also contribute to the rate of Mn attachment on the microbe? Yes but to a very small extent which did not affect the results presented.

Comment 28: Figure 6: why did you present only 4% soil, how about 6% that produced highest biosurfactant in the microbes? The answer to this question is written above and highlighted.

Conclusion

Comment 29: I suggest the opening sentence be delete since you did not work on different soils and the effects of soil conditions on the microbe(s). The sentence was deleted (line 407-408 in the revised manuscript)

Comment 30: line 400: "applied" not "developed". The sentence was revised according to the Reviewer’s suggestion (line 406 in the revised manuscript).

Comment 31: line 403: delete "simultaneous" The word was deleted (line 410 in the revised manuscript).

 

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