Fenton-Mediated Chlorophenol Degradation by Iron-Reducing Compounds Isolated from Endophytic Fungi in Atacama Puna Plateau Lecanicillium ATA01
, Paris Lavin 4, Cristina Purcarea 5, Manuel Alarcón 2, Danae Irribarren-Riquelme 6 and Jaime Rodríguez 7Round 1
Reviewer 1 Report
Introduction is lengthy, may be reduced.
Too many citations are useless. Citations in a research must not exceeds than 40. There are too old citations as well.
Author Response
REVIEWER #1
Dear Reviewer,
First of all, we are grateful for the general and specific comments, suggestions and the constructive comments given by yourself, which have substantially improved our manuscript. Specific responses to each of the comments are set out below.
Basically, we reduce the extensión of section “Introduction” by removing specific parts. In this new version (attached as new MS), we moved all the data or quantitive information to the section “Results”, we removed results and any data from the section “Discussion” and finally focused and synthetized our statements in the section “Conclusions”. Regarding the use of English language, we modified and remove several sentences and parragraphs to keep the main messages simple as possible.
Comments and Suggestions for Authors
Point #1 - Introduction is lengthy, may be reduced.
The length of introduction was reduced from 2,5 pages to 2 pages (in the new MS versión, attached)
Point # 2- Too many citations are useless.
Point # 3 - Citations in a research must not exceeds than 40.
Point # 4- There are too old citations as well.
We made a considerable efforts to reduce the number of citations by removing useless references along the main text (Point #2). Regarding old citations (Point #4), we decided to update all the references and establish a cut-off in the year 2000, removing the rest of old citations (certainly there are some exception specially in Material and Method, where we maintain some references). Despite of you suggestion to reduce the number of references to 40, we did our best effort, trying to do not alter the structure of the research article, and we reduced significantly the number of citations from 106 to 47 references. We sincerely hope this reduction is considered hepful to improve the new verison MS.
Author Response File: 
Author Response.pdf
Reviewer 2 Report
The work presented here is not particularly interesting, firstly, because of the way the article is structured, and secondly, because of the poor use of language. Regarding the first aspect, the Introduction includes data, the Discussion includes results and the Conclusions discusses them. Regarding the second aspect, the level of English can be improved, as can the use of punctuation marks, which sometimes makes it very difficult to interpret sentences. On the other side, terms are repeated in the same paragraph. Some examples:
- The compounds not only had the ability to chelate and reduce 24
Fe+3 to Fe+2 but also, as ferric IRC complex oxidized recalcitrant lignin model substrates such as veratryl alcohol (VA), 2,6-dimethoxyphenol (DMP), and 2,2’-azino-bis (3-ethylbenzothiazoline-6-sulphonic acid (ABTS) in the presence and absence of hydrogen peroxide. - Furthermore, in the same iron concentration...
- The highly toxic and persistent environmental pollutants chlorophenol (CPs) compounds (and derivatives) are commonly used in the manufacture of industrial products such as dyes, drugs, pesticides, thus can be found...
- Notwithstanding several physicochemical and biological methods that have been used for the removal of CPs from the environment 5 microbial degradation has been considered a cost-effective and eco-friendly approach to remove CPs from the environment.
- The wood degradation mechanisms displayed by white and brown rot fungi, besides enzymes, involve the activity of low molecular mass oxidants between them lignin peroxidase LiP mediators, organic acids Mn+3 chelates, laccase mediators, peroxyl radicals produced from lipid peroxidation and iron-reducing compounds (IRC) (the hydroxyl radical production via Fenton chemistry, eq.1) has been postulated.
- Rodriguez and collaborators 38 analyzed different examples of Fenton reaction is driven hydroxybenzenes and its application in the oxidation of lignin and a pulp bleaching effluent mediated by 2,3-dihydroxybenzoic and 3,4-dihydroxyphenylacetic acid as well as the reduction of Fe+3 to Fe+2 mediated by catechol–iron complexes and subsequent oxidation of o-dianisidine.
- According to this, the endophytic fungi Lecanicillium ATA01 (Ascomycota, Hypocreales), isolated from extreme environment Atacama Puna Plateau, was selected as target specie for the tests due to play additional roles in nature as plant disease antagonism and plant colonization and growth promotion due to rhizosphere colonization.
- k = logn ((C0-C1) +1)/logn(t), were C0 corresponds...
- ...the increase in absorbance at 270 nm due malonate-Mn+3 complex formation...
- The fraction A reacted strongly with CAS reagent, Arnow, Bradford and Ferrozine tests however Csáky iron chelant test and Miller test were negative.
- Lecanicillium sp. ATA01 is recognized as entomopathogenic endophytic able to colonize...
- ...encouraging nutrient acquisition, increase reproductive success, conferring biotic defense or abiotic stress tolerance to the host plant.
Other suggestions:
- Thermal values must be separated from the corresponding units.
- The need to characterize the morphological and microscopic properties of the isolate, when molecular identification clears any doubt about its identity, is not fully understood.
- Why are most of the references included in M&M not in numerical format?
- The capacity to reduce Fe+2 to Fe+3... ¿reduce?
- In Figure 6, the Y-axis label is incorrect. It should be "Remanent concentration".
Author Response
REVIEWER #2
Dear Reviewer,
First of all, we are grateful for the general and specific comments, suggestions and the constructive comments given by yourself, which have substantially improved our manuscript. Specific responses to each of the comments are set out below. Basically, we reduce the extensión of section “Introduction” by removing specific parts. In this new version (attached as new MS), we moved all the data or quantitive information to the section “Results”, we remove results and any data from the section “Discussion” and finally focused and synthetized our statements in the section “Conclusions”. Regarding the use of English language, we modified and removed several sentences along the main text to keep the main messages simple as possible.
Comments and Suggestions for Authors
The work presented here is not particularly interesting, firstly, because of the way the article is structured, and secondly, because of the poor use of language. Regarding the first aspect, the Introduction includes data, the Discussion includes results and the Conclusions discusses them. Regarding the second aspect, the level of English can be improved, as can the use of punctuation marks, which sometimes makes it very difficult to interpret sentences. On the other side, terms are repeated in the same paragraph. Some examples:
Point # 1. The compounds not only had the ability to chelate and reduce 24
Fe+3 to Fe+2 but also, as ferric IRC complex oxidized recalcitrant lignin model substrates such as veratryl alcohol (VA), 2,6-dimethoxyphenol (DMP), and 2,2’-azino-bis (3-ethylbenzothiazoline-6-sulphonic acid (ABTS) in the presence and absence of hydrogen peroxide.
In lines L24-L27, a modified parragraph was incorporated as follows: “These compounds were able to chelate and mediate reduction of Fe+3 to Fe+2 and oxidized recalcitrant lignin model substrates such as veratryl alcohol (VA), 2,6-dimethoxyphenol (DMP), and 2,2’-azino-bis (3-ethylbenzothiazoline-6-sulphonic acid (ABTS) with or without hydrogen peroxide.”
Point # 2. - Furthermore, in the same iron concentration...
In lines L29-L31, the sentence “…in the same iron concentration..” was removed and new phrase was structured accordingly: “Furthermore, Fenton-like reactions using the synthetic iron chelates DTPA and EDTA and free Fe+2 and Fe+3 were also carried out to compare with the reaction mediated by IRCs.”
Point #3 - The highly toxic and persistent environmental pollutants chlorophenol (CPs) compounds (and derivatives) are commonly used in the manufacture of industrial products such as dyes, drugs, pesticides, thus can be found...
In line L39-L42, the original phrase was modified and basically split it in two according to this structure: “The highly toxic and persistent environmental pollutants chlorophenol (CPs) compounds (and derivatives) are commonly used in the manufacture of industrial products such as dyes, drugs and pesticides. The excessive use of these industrial products and their discharge in effluents have produced the contamination of soil and waters [1]. “
Point #4 - Notwithstanding several physicochemical and biological methods that have been used for the removal of CPs from the environment [5] microbial degradation has been considered a cost-effective and eco-friendly approach to remove CPs from the environment.
In lines 48-50, modifications to the original parragraph were incorporated as follows: “Several physicochemical and biological methods that have been used for the removal of CPs from the environment [3] however, microbial degradation techniques still are considered an interesting cost-effective and eco-friendly approach [1].”
Point #5 - The wood degradation mechanisms displayed by white and brown rot fungi, besides enzymes, involve the activity of low molecular mass oxidants between them lignin peroxidase LiP mediators, organic acids Mn+3 chelates, laccase mediators, peroxyl radicals produced from lipid peroxidation and iron-reducing compounds (IRC) (the hydroxyl radical production via Fenton chemistry, eq.1) has been postulated.
In lines L60-64, the original parragraph was changed as follow: “In this sense, wood decay mechanisms displayed by white and brown rot fungi, besides enzymes, involve the activity of low molecular mass oxidants such as lignin peroxidase LiP mediators, organic acids Mn+3 chelates, laccase mediators, peroxyl radicals produced from lipid peroxidation and iron-reducing compounds (IRC) (the hydroxyl radical production via Fenton chemistry.”
Point #6 - Rodriguez and collaborators [38] analyzed different examples of Fenton reaction is driven hydroxybenzenes and its application in the oxidation of lignin and a pulp bleaching effluent mediated by 2,3-dihydroxybenzoic and 3,4-dihydroxyphenylacetic acid as well as the reduction of Fe+3 to Fe+2 mediated by catechol–iron complexes and subsequent oxidation of o-dianisidine.
In lines L89-90, the phrase was slightly modified “Rodriguez and collaborators [15] analyzed different examples of Fenton reaction driven by hydroxybenzenes and its application in the oxidation of lignin and a pulp bleaching effluent mediated by 2,3-dihydroxybenzoic and 3,4-dihydroxyphenylacetic acid as well as the reduction of Fe+3 to Fe+2 mediated by catechol–iron complexes and subsequent oxidation of o-dianisidine.
Point #7 - According to this, the endophytic fungi Lecanicillium ATA01 (Ascomycota, Hypocreales), isolated from extreme environment Atacama Puna Plateau, was selected as target specie for the tests due to play additional roles in nature as plant disease antagonism and plant colonization and growth promotion due to rhizosphere colonization.
In lines L111-113, the sentence was modified as follows: “According to this, the endophytic fungi Lecanicillium ATA01 (Ascomycota, Hypocreales), isolated from the extreme environment Atacama Puna Plateau, was selected as the target species for chlorophenol degradation.”
Point #8 …k = logn ((C0-C1) +1)/logn(t), were C0 corresponds...
In lines L262-264; section 2.9 “Rate constant and yield of chlorphenol degradation by Fenton-like reaction”; a new expression was incorporated… “k = logn ((C0-C1) +1)/logn (t), were C0 corresponds to the initial percentage of chlorophenol at the beginning reaction in time 0; C1 correspond to the percentage of chlorophenol”
Point #9 - ...the increase in absorbance at 270 nm due malonate-Mn+3 complex formation...
In lines L200-201, the sentence was corrected as follows:…”was measured by monitoring the increase in absorbance at 270 nm due malonate-Mn+3 complex formation”
Point #10 - The fraction A reacted strongly with CAS reagent, Arnow, Bradford and Ferrozine tests however Csáky iron chelant test and Miller test were negative.
In lines L310-312, the original phrase was corrected according to this phrase: “The fraction A obtained marked positive reaction in CAS, Arnow, Bradford and Ferrozine assays however, Csáky and Somogyi-Nelson assays were negative”.
Point #11 - Lecanicillium sp. ATA01 is recognized as entomopathogenic endophytic able to colonize...
In lines L453-456, the original phrase was changed as follows: “Lecanicillium sp. ATA01 is an entomopathogenic endophytic fungus isolated from the extreme environment natural laboratory Atacama Puna Plateau and consequently well-adapted to stressful conditions (i.e., cold temperatures, drought, high radiation, poor soils)”
Point # 12...encouraging nutrient acquisition, increase reproductive success, conferring biotic defense or abiotic stress tolerance to the host plant.
In lines L453-456, the original phrase was modified. Indeed, this phrase “encouraging nutrient acquisition, increase reproductive success, conferring biotic defense or abiotic stress tolerance to the host plant” was removed completely to avoid confusión.
Other suggestions:
- Point #13 - Thermal values must be separated from the corresponding units.
All themal values along the text were separated from the corresponding units
- Point #14 - The need to characterize the morphological and microscopic properties of the isolate, when molecular identification clears any doubt about its identity, is not fully understood.
We do not fully agree fully with this sentence. For molecular identification of fungal species, the best strategy is the sequencing of múltiple genes (multi-locus approach). When you do not have this complete information and when your database of sequences are not curated it is recommended to include and classical taxonomical information. According to Gautam and collaborators, the available information on classical and modern methods compiled revealed that the DNA based molecular studies are still scant, and more studies are required to achieve the accurate estimation of fungi (Gautam, A. K., Verma, R. K., Avasthi, S., Bohra, Y., Devadatha, B., Niranjan, M., & Suwannarach, N. (2022). Current Insight into Traditional and Modern Methods in Fungal Diversity Estimates. Journal of Fungi, 8(3), 226. That is the reason that in this work our option was include and complement the molecular identification at genus level using classical taxonomic approach.
- Point #15 - Why are most of the references included in M&M not in numerical format?
All the references in M&M are in numerical format accordingly
- Point #16 - The capacity to reduce Fe+2 to Fe+3... ¿reduce?
In L308-310; the original phrase was modified as follows: “The reduction ability Fe+3 to Fe+2 as well as the oxidation of substrates were found mainly in fraction A while fraction B did not react significantly.”
- Point #17 - In Figure 6, the Y-axis label is incorrect. It should be "Remanent concentration".
All Y-axes in figure 6 were changed. The expression “Remanent concentration of…” was included (see page 11 in the new MS versión - attach
Round 2
Reviewer 2 Report
The authors have implemented certain improvements that have increased the quality of the article. By the way, it would be advisable for future occasions that, in addition to including the changes in the responses to the reviewers, they highlight them in the text.
Although the level achieved does not make this article a leading article, it does allow it to show the minimum required. However, there are still some small details that can be corrected.
The use of punctuation is still sometimes inadequate, making sentences excessively long and difficult to understand (L 89-93; L-103-106; L. 461-464; L. 522-525).
Please review section 2.9. The definition of parameters C0 and C1 is the same and, on the other hand, two definitions of C1 are given.
L. 49: Include punctuation before “however”.
L.51: Change comma from still to topic.
L. 67: Close parenthesis.
L. 181: Replace was with were.
L. 260: Replace were with where.
L. 283: Replace the comma with a period.
L. 289: Replace is with are.
L. 309: Replace however, with ,while.
L. 314: Delete of.
L. 330: Replace however with although.
L. 353: Delete period after 100 mM and and puti t after occur.
L. 357: Replace period with comma or change Whereas by On the contrary.
L. 366: Replace to with for.
L. 456: Replace As far with As far as.
L. 498: Put reference 43 into brackets.
L. 499: Delete comma.
L. 501: Include comma before as well.
L. 502: Replace products with product.
Author Response
RESPONSE TO REVIEWER COMMENTS
Dear Mr/Ms Reviewer,
First, we are grateful for the constructive comments given by yourself, which have substantially improved our manuscript. Specific responses to each of the comments are set out below.
Reply Comments and Suggestions for Authors
The authors have implemented certain improvements that have increased the quality of the article. By the way, it would be advisable for future occasions that, in addition to including the changes in the responses to the reviewers, they highlight them in the text.
Although the level achieved does not make this article a leading article, it does allow it to show the minimum required. However, there are still some small details that can be corrected.
The use of punctuation is still sometimes inadequate, making sentences excessively long and difficult to understand (L 89-93);
REPLY. The following paragraph was divided in two main sentences. Specifically, in L91 of the new MS version, a new sentence was inserted. "Rodriguez and collaborators 15 analyzed different examples of Fenton reaction driven by hydroxy benzenes and its application in the oxidation of lignin and a pulp bleaching effluents. The catechol-compounds 2,3-dihydroxybenzoic and 3,4-dihydroxyphenylacetic acid were used to mediate reduction from Fe+3 to Fe+2. Morever, these compounds as catechol–iron complexes were able to promote the subsequent oxidation of o-dianisidine."
...L-103-106;
REPLY. The following paragraph was divided in two main sentences. Specifically, in L106, a new sentence was inserted. "Lecanicillium species belongs to a group of entomopathogenic fungi and are among the most studied and virulent fungi related to the order Hypocreales (Fungi: Ascomycota). Previous reports have been recognized their ability to secrete cell wall-degrading enzymes such as chitinases and lipases [18] as well as extracellular Mn-peroxidase [19] useful during the decomposition of complex biopolymers."
...L461-464;
REPLY. In L463-466 the structure of paragraph was included according to "The degradation of 2,4-DCP by Lecanicillium species was previously reported as a laboratory viable option 38. The degradation of anthracene (40 %), phenanthrene (63 %), and fluorine (81 %) within 14 days as well as the production of extracellular peroxidases stimulated by Mn2+ was detected in Lecanicillium aphanocladii 38.
..L522-525.
REPLY. Added in L524, a new structure was incorporated as follows: "There is an increasing interest in the study of the genus Lecanicillium sp. as biological decomposer of harmful compounds mainly because of the enzymatic mechanisms involved in the degradation of oil and some PAHs [19]."
Please review section 2.9. The definition of parameters C0 and C1 is the same and, on the other hand, two definitions of C1 are given.
REPLY. Added in L263-267, a new structure of the paragraph was inserted, just one single definition of C1 is now provided. "...the following equation: k = logn ((C0-C1) +1)/logn (t), where C0 corresponds to the initial percentage of chlorophenol at the beginning reaction in time 0; C1 corresponds to the percentage of chlorophenol in the reaction mixture at the end the logarithmic slope (t = end time logn in h)."
L49: Include punctuation before “however”.
Reply. modification included in (L49)
L.51: Change comma from still to topic.
Reply. modifcation incorporated in (L51)
L67: Close parenthesis.
Reply. modification incorporated in (L67)
L181: Replace was with were. Reply, replaced in (L180)
L260: Replace were with where. Reply, replaced in (L263)
L283: Replace the comma with a period. Reply, replaced in (L286)
L289: Replace is with are. Reply, replaced in (L292)
L309: Replace however, with, while. Reply, replaced in (L311)
L314: Delete of. Reply, deleted in (L317)
L330: Replace however with although. Reply, incorporated in (L334)
L353: Delete period after 100 mM and and puti t after occur. Reply, incorporated in (L357)
L357: Replace period with comma or change Whereas by On the contrary. Reply, incorporated in (L381, it was added the expresion.. “On the contrary”
L366: Replace to with for. Reply, it was replaced in (L370)
L456: Replace As far with As far as. Reply, it was replaced in (L460)
L498: Put reference 43 into brackets. Reply, modification incorporated in (L504)
L499: Delete comma. Reply, modification incorporated in (L505)
L501: Include comma before as well. Reply, modification incorporated in (L507)
L502: Replace products with product. Reply, modification incorporated in (L508)
Author Response File: Author Response.pdf
