Adsorption of Salmonella in Clay Minerals and Clay-Based Materials

Round 1

Reviewer 1 Report

Dear author,

The manuscript presents an interesting subject related with the use of some clay minerals and clay-based materials to understand the adsorption behavior of salmonella, but several improvements must be made:

- The “1. Introduction” section is too long and could be reduced;

- The aim and goals of the research are too much wide and broad, since the characterization of the studied clay minerals (sepiolite, montmorillonite, palygorskite and saponite) was already published in several publication;

- The section “3.1. Characterization of the starting clay…” is too much long (with 10,5 pages of text) and too much descriptive. The main goal of the research – “3.2. Adsoption of salmonella testes” section has only almost 4 pages of text. This proportion is incoherent concerning the focus of the research;

- Some figures can be deleted (ex. Figure 1) since the patterns are repeated in figures 2 and 3. Some figures (ex. Figure 4 and 5) must be merged since report the same subject;

- Some porous/porosity consideration could not be “clearly” taken from SEM, since some porosity techniques (ex. N2-adsorption) were used;

- In general, the English must be improved, since from times to times, it is missing points, commas, incorrect paragraphs, some sentences are repeated (ex. in 3.1 section), some sentences are confused and must be rewritten, units written different in different sections of the manuscript;

- The 3.2 section do not present interpretation, correlation and corroboration of the obtained behavior, based in the data showed in section 3.1. This is a weakness of the manuscript that must be improved.

- In section 3.2 there are tables and figures which present the same information. But it is missing some deep explanation and “possible” clues concerning the Salmonella adsorption behavior. Another weakness of the manuscript is related with the absence of linear correlations and simply cluster analysis between the values obtained in characterization (section 3.1) and the values obtained in adsorption (section 3.2). Once, what was idea in having a long characterization section, if the values obtained in this characterization are not used to deeply explain the Salmonella adsorption behavior?;

- Several (too much) values presented in the text (ex. FTIR and adsorption) are different in what concern the same values presented in figures (ex. FTIR) or tables (Table 2). All the numbers in text must be checked number by number in all manuscript.

- It is missing some methodological information about DFT method for pore size distribution;

- Some references are incorrect inserted in the paragraph;

- Some figures (ex. figure 16) present wrong title;

- Conclusions present values and units that were not showed in previous sections.

The attached file presents (inside yellow sticky notes) the comments/suggestions that were annotated in the manuscript.

Comments for author File: Comments.pdf

Author Response

Reviewer 1

Authors are very grateful to the reviewer 1 whose suggestion have notably improved the work. then I show you the answer to the review:

The manuscript presents an interesting subject related with the use of some clay minerals and clay-based materials to understand the adsorption behavior of salmonella, but several improvements must be made:

- The “1. Introduction” section is too long and could be reduced;

According to the suggestion of R-1 Introduction section has been reduced

- The aim and goals of the research are too much wide and broad, since the characterization of the studied clay minerals (sepiolite, montmorillonite, palygorskite and saponite) was already published in several publications;

According to the suggestion of R-1 the aim and goals have been modified

- The section “3.1. Characterization of the starting clay…” is too much long (with 10,5 pages of text) and too much descriptive. The main goal of the research – “3.2. Adsoption of salmonella testes” section has only almost 4 pages of text. This proportion is incoherent concerning the focus of the research;

The main goal of this work is to evaluate the capacity of some clay mineral with different physicochemical properties and chemical compositions to retain salmonella from water with the aim of find a relationship between these characteristics and the effectiveness of the sorption process. For this reason, in a first section of the paper is focused on the highlighting of the differences between crystalochemical characteristics of the starting clay minerals. Authors opinion is that this section is necessary and appropriate for the topic of this special issue for the journal “MINERALS”.

As the reviewer have indicated, the adsorption data can be relatively short. However, these data are preliminary results for subsequent studies, where we are going to incorporate some transition metal to favour the adsorption and degradation of this pathogen.

- Some figures can be deleted (ex. Figure 1) since the patterns are repeated in figures 2 and 3. Some figures (ex. Figure 4 and 5) must be merged since report the same subject;

According to the suggestion of R-1 we removed Figure 1 . Nevertheless, Figure 4 and 5 have not been merged to facilitate the visualization of the readers. On the other hand, the authors have also merged the N₂ adsorption isotherms and pore size distribution, as suggested the reviewer.

- Some porous/porosity consideration could not be “clearly” taken from SEM, since some porosity techniques (ex. N2-adsorption) were used;

According to the suggestion of R-1 the SEM section has been modified

- In general, the English must be improved, since from times to times, it is missing points, commas, incorrect paragraphs, some sentences are repeated (ex. in 3.1 section), some sentences are confused and must be rewritten, units written different in different sections of the manuscript;

According to the suggestion of R-1, the manuscript has been completely reviewed by a professional native English translator

- The 3.2 section do not present interpretation, correlation and corroboration of the obtained behavior, based in the data showed in section 3.1. This is a weakness of the manuscript that must be improved. In section 3.2 there are tables and figures which present the same information. But it is missing some deep explanation and “possible” clues concerning the Salmonella adsorption behavior. Another weakness of the manuscript is related with the absence of linear correlations and simply cluster analysis between the values obtained in characterization (section 3.1) and the values obtained in adsorption (section 3.2). Once, what was idea in having a long characterization section, if the values obtained in this characterization are not used to deeply explain the Salmonella adsorption behavior?;

Following the suggestion of R-1 we included a new figure (Fig. 14) and a additional discussion of the results:

“Figure 14. Salmonella adsorption capacity variations vs external surface of the sorbent

The results of this initial study show that all the materials analyzed have great salmonella adsorption capacities ranging from the lowest value observed in the mont-PCH sample (0.29 x10¹⁰ CFU g^-1) to the highest value observed in the natural palygorskite sample (1.52 x10¹⁰ CFU g^-1). However, the variations of this capacity between the starting materials and their derived seems to be difficult to explain because these erratic variations do not show clearly defined trends (Fig. 14).

An overview of the results suggests that the structural characteristics of the starting clays do not appear to be the controlling factor for salmonella adsorption capacity. Thus, for example, smectite group minerals having very similar layered structures with an expandable interlayer space show very different adsorption capacities. However, given the large size of the Salmonella cells, in many cases larger than the size of the individual montmorillonite or saponite particles, it is unable to enter the interlayer space. Therefore, the adsorption of salmonella must occur mostly on the available external surface of aggregates of laminar particles of saponite or montmorillonite. For this reason, in the case of the smectite group minerals studied, the Salmonella adsorption capacity increases with the available external surface. Nevertheless, the availability of adsorption centers present on this external surface is strongly influenced by the volume of the pores. The adsorption centers located inside the micropores and nanopores are inaccessible due to the large size of the salmonella cell. For this reason, other things being equal, the Salmonella adsorption capacity seems to depend on the external surface exposed in macropores of adequate size or on the outermost external surface of the aggregates of laminar particles. The smallest particle size of the natural saponite sample, observed by SEM (Fig. 3), and its less compact distribution are the factors that lead to a greater adsorption capacity compared with that of montmorillonite.

On the other hand, we also find notable differences in the adsorption capacities of fibrous phyllosilicates: sepiolite and palygorskite. In this case, although the sepiolite has an external surface area (135 m² g^-1) significantly greater than that of the palygorskite (78 m² g^-1), SEM study showed that the fibers morphologies of both minerals are similar. Since the cross-section of the structural channels of sepiolite and palygorskite are significantly smaller than the size of the Salmonella cells, it must only be adsorbed on the external surface of the fibers. For this reason, it would be expected that sepiolite, with a larger external surface than palygorskite, has a greater capacity for adsorption of Salmonella. Nevertheless, the opposite effect is observed. The Salmonella adsorption capacity of natural palygorskite is greater than that of sepiolite. In this case, in addition to the availability of the adsorption centers of the external surface of both minerals, we must take into account other characteristics such as the chemical nature of this external surface. Most of the adsorption mechanisms of these clay minerals are usually based on ion exchange reactions that preferentially adsorb cations. However, the Salmonella cells, like most gram-negative organisms, has a net negative charge [57]. This suggests that the interaction of salmonella with the external surface of the clay particles can occur through other types of active centers, such as silanol groups or structural defects in the grain edges generating charge deficits. The palygorskite usually has a large number of silanol groups on its external surface compared to the sepiolite [58], and this chemical characteristic controls the greater capacity of the palygorskite to retain salmonella.

The synthesis of PCH with the different clay minerals has a very diverse influence on the Salmonella adsorption capacities. In general, the textural modifications caused by the synthesis lead to the formation of new external surface (ranging between 100 and 168 m² g^-1) and to the formation of a large number of micropores whose small sizes makes them inaccessible to Salmonella cells. Much of the generated external surface is inside of those micropores so it is unable to adsorb Salmonella. Thus, for example, in the samples with greater increases in external surface, (saponite ΔSext = 165 m² g^-1 or palygorskite ΔSext = 168 m² g^-1), the synthesis of PCH does not produce an increase in the adsorption capacity. This result suggests that the new generated surface does not have appropriate adsorption centers to retain Salmonella cells. On the other hand, in the montmorillonite sample, the synthesis of PCH causes a decrease in Salmonella adsorption capacity from 0.50 x10¹⁰ to 0.29 x10¹⁰ CFU g^-1 although there is an increase in external surface area of ​​109 m² g^-1. This result suggests that some adsorption centers of the starting montmorillonite are possibly inactivated by an excess of reagent that crystallizes on the montmorillonite-PCH particles. However, a different behavior shows the sepiolite-PCH sample. In this case, the synthesis of PCH produces an increase in the capacity of adsorption of salmonella from 0.46 x10¹0 to 1.06 x10¹⁰. This increase seems to be related to the separation of fiber bundles that occurred during the synthesis of PCH observed in SEM micrographs (Fig. 4). This separation made accessible new adsorption centers that were previously located inside the packages.”

- Several (too much) values presented in the text (ex. FTIR and adsorption) are different in what concern the same values presented in figures (ex. FTIR) or tables (Table 2). All the numbers in text must be checked number by number in all manuscript.

According to the R-1 advices we made all corrections along the manuscript

- It is missing some methodological information about DFT method for pore size distribution;

The DFT method is a data provided by the ASAP 2020 equipment. This data is better in comparison to other equations to determination of the pore size distribution due to other equation, such as BJH, are only well fitted for micro- and mesoporous materials. The information and reference is included in the experimental section

- Some references are incorrect inserted in the paragraph;

Following the suggestion of R-1 the references has been revised and some of them have been deleted

- Some figures (ex. figure 16) present wrong title;

Fig. 16 renamed as Fig 13 has been corrected

- Conclusions present values and units that were not showed in previous sections.

Author Response File: Author Response.pdf

Reviewer 2 Report

In the present manuscript the authors report the use of four different clays and their modified analogues as adsorbent for Salmonella. The aim of the work is novel and noteworthy, although there are some issues which shloud be addressed before publication on Minerals.

There are too many typos ad grammatical mistakes in the manuscript. Please carefully revise and make improvements. See for example pag 5 line 206, pg 6 line 222 and so on. Please revise the abstract. The manuscript is mainly focused on the physico-chemical characterizations of the four clays and modified ones. Please add some consideration about this in the abstract as well as in the introduction and conclusions part. In my opinion the authors should better emphasize the need to modify the clays in order to obtain good adsorbent materials.

Author Response

Reviewer 2

Authors are very grateful to the reviewer 2 whose suggestion have notably improved the work. then I show you the answer to the review:

-There are too many typos ad grammatical mistakes in the manuscript. Please carefully revise and make improvements. See for example pag 5 line 206, pg 6 line 222 and so on. Please revise the abstract.

According to the suggestion of R-1, the manuscript has been completely reviewed by a professional native English translator

-The manuscript is mainly focused on the physico-chemical characterizations of the four clays and modified ones. Please add some consideration about this in the abstract as well as in the introduction and conclusions part.

The abstract has been modified according the reviewer 2 as follows:

“A series of clay minerals and clay-based materials have been tested to eliminate one of the most dangerous bacteria we can find in the water: Salmonella. It has been proven that the use of clays and their PCH materials can be a suitable method for removing Salmonella from water. The results of this initial study show that all the materials analyzed have great salmonella adsorption capacities ranging from the lowest value observed in the mont-PCH sample (0.29 x10¹⁰ CFU g^-1) to the highest value observed in the natural palygorskite sample (1.52 x10¹⁰ CFU g^-1). Macroporosity, accessible external surface area, and the presence of silanol groups in the external surface of the particles appears to be the controlling factors for Salmonella adsorption capacity while it seems that the structural characteristics of the clay minerals and their respective PCH does not affect the adsorption capacity.”

-In my opinion the authors should better emphasize the need to modify the clays in order to obtain good adsorbent materials.

With the results obtained in this work we know the factor controlling the salmonella adsorption capacity. From this study we can redirect the synthesis towards the formation of materials with an appropriate macroporosity in order to improve the adsorption of salmonella cells

Author Response File: Author Response.pdf

Round 2

Reviewer 1 Report

Dear authors,

The manuscript was improved concerning the sent comments and suggestions, but some corrections must be made:

- In general, the English was improved, but there are some sentences were the text must be rewritten;

- In the “Introduction” section, the text concerning the toxicity of the palygorkskite must be changed, since this mineral cannot be classified as non-toxic.

- The section “Adsoption of salmonella tests” still presents some mistakes concerning the values referred in the text and presented in table 1 and 2.

The attached file presents (inside yellow sticky notes) the comments/suggestions that were annotated in the manuscript.

Comments for author File: Comments.pdf

Author Response

The authors are very grateful to the great work of the reviewer who has undoubtedly improved the article significantly. Below we detail the responses to the reviewer's comments:

Where is reference [7] in the text?

The authors removed the reference number 7 wrong. This error was solved by the authors.

According reference Devlop. Clay Science 2011 (Chapter 11) palygorskite could be dangerous for human health, since cannot be classified as non-toxic.

The use of fibrous clays seems to generate controversy. In the opinion of the authors of the work, the fibers are large enough to cause health problems. However, the authors will indicate that the effects of these phyllosilicates on health depends on their physicochemical properties.

“In some cases, the use of these fibrous phyllosilicates has generated controversy due to the carcinogenic activity of these minerals seems to be mostly dependent on their physico-chemical properties (silanol groups), crystallinity and fibre length, which are occasionally related to their genetic environment. However, their potentially hazardous effects should be assessed individually for each deposit and factory on the basis of epidemiological data and the results of sensitive animal tests.”

Again, parenthesis after parenthesis...

The authors have corrected this sentence “(TSA), (Oxoid Ltd., Basingstoke, UK)”

In the same paragraph "hours" and "h"

The authors have written hours in both place

"Grados" was not changed.... Please, in caption figure, insert all acronyms that are presented in figure 2.

The x-axis was corrected and the acronyms of reported in figure 2 appear in its list of caption now.

Missing "," (Figure 5 and Figure 6)

The authors have added this comma in the list of caption of Figure 5 and Figure 6.

After "method" insert "(Table 1)"

Added.

As in indicated for microporosity (line 417), after "mesoporosity" and "macroporosity" insert between parenthesis the range size for both in nm.

The authors have rewritten the paragraph, adding the suggestion of the reviewer.

“In the case of Mont and Sap, the mesoporosity (2-50 nm) is relatively low. However, both Sep and Pal display a high proportion of meso- and macroporosity (> 50 nm) as indicates the growing of the N₂-adsorbed at high relative pressure due to the voids between fibers”.

"p" - lower case (line 438).

Corrected.

Atention: I have not said "the adsorption data can be relatively short ". I said exactly the opposite. And maintain my opinion. This section is still very large (app. 8 pages), concerning the aim of the study and also the referred in the title of the manuscript, which do not fill nothing related to the characterization of the clay samples and respective PCH.

Why change what was correct for incorrect? "m" must be "M" - upper case "s" must be "S" upper case. In these two cases the first letter of a sentence is upper, not lower case.

The authors have corrected these terms.

This paragraph (lines 480 to 483) is outside any context.

Following the advice of the reviewer, the paragraph “These differences in adsorption kinetics show that in the PCH, Salmonella is adsorbed in the most easily accessible places, whereas in the case of natural montmorillonite, a small proportion of Salmonella (5 %) can be retained in more inaccessible positions, therefore requiring longer contact time”.

Repeated in line 487 (Fig.9, grey line)

This term has been removed in Fig. 488.

Rewrite: "The Salmonella capacity of montmorilonite", line 503

Corrected. This data has been corrected in following section in the manuscript.

Line 504, As this is the first time were is referred g-1, it will be interesting, write here how is made this conversion.

We included the following paragraph in the 2.4. (Preparation of treatment solutions and sorption experiments) section

“The salmonella adsorption capacity, measured in colony forming units per gram of clay (CFU g-1), is calculated by means the difference of Salmonella concentrations between the control and the sorbents-treated solutions, taking into account that 4 mg of sorbents were added to 50 ml of solution.”

Line 517,Please, pay attention on the values presented in text: 0.144 is for 4 hours, not 1 hour....the correct is 0.120.

Corrected

Line 518, What is "both cases"? This is only valid for saponite.

Corrected

Line 521."0.06" or "around 0.06" or "approximatly 0,06"? saponite is 0.066 and sap-PCH is 0.063

Following the suggestion of the reviewer, the sentence has been rewritten. “the absorbance continues decreasing in the next hour, with both saponite and saponite-PCH sorbents, at a noticeably lower rate until to values of around 0.06 in both cases”.

Line 527, If the sites are "inaccessible", how can adsorb salmonella. Please, rewrite this conclusion in correct scientifc context.

As indicates the reviewer, the sentence “These results suggest that there is a strong adsorption of Salmonella in the first hour of contact, especially in the most accessible surfaces such as the external surface and in the pores of greater size, which is followed by a small additional adsorption after two hours of contact in which Salmonella could occupy some more inaccessible sites.” Causes confusion to the readers so this sentence has been completed as indicates in the next paragraph”

“These results suggest that there is a strong adsorption of Salmonella in the first hour of contact, especially in the most accessible surfaces such as the external surface and in the pores of greater size, which is followed by a small additional adsorption after two hours of contact in which Salmonella could occupy some more inaccessible sites located on the edge of the sheets. The insertion in the porous structure or within interlayer sheet must be discarded”

Line 544 “"approximately" is before 0.7”

Corrected

Line 545 “This value (10^5.94)is for 1 hour? This must be referred in text.

According the suggestion of the reviewer, this information has been incorporated into the sentence “the concentration from 10^6.09 to 10^5.67 CFU mL^-1 after the first hour”

Line 580, This is incorrect. The correct is 0.112 As suggested in the first review, please, review all these values in this section and compare them with the indicated in table 2.

As indicates the reviewer, this value has been corrected. In addition, the data reported in the manuscript have been compared with those reported in Table 2.

Line 592, Table 1 do not presents size but volume, and also palygorskite presents the lowest Vmicrop (0,0060 cm3 g-1)

We have taken into account the correct comment of the reviewer and have modified the paragraph as follows:

Differences in capacity of Salmonella adsorption may be due to the presence of a greater number of broken bonds or silanol groups on the external surface of the natural palygorskite, which can favors the electrostatic interaction with the surface of the Salmonella.

The Salmonella adsorption capacity of palygorskite decreases with the synthesis of the PCH:

palygorskite (1.52 x10¹⁰ CFU g-1) > pal-PCH (1.48 x10¹⁰ CFU g^-1)

Author Response File: Author Response.docx