Metabolic Biodegradation Pathway of Fluoranthene by Indigenous Trichoderma lixii and Talaromyces pinophilus spp.

Round 1

Reviewer 1 Report

This work describes the metabolic pathway of fluoranthene degradation by a couple of fungal isolates by extracellular enzymes, the work is well presented, in logical and consistent order. However, there are several points that will substantially improve the quality of the manuscript, which are:

 

Major comments

 

The identification of the strains requires a thorough analysis to confirm if the strains correspond to the fungal genera mentioned in the manuscript. This is relevant since both fungal strains are mentioned from the title of the paper, it is only slightly commented (a single line) in the materials and methods section, but it leaves doubts because the accession number is the same for both strains. In addition, making a simple blast of the mentioned sequence (MK208694) displays other fungal genera. Therefore, it is necessary to include a more solid characterization of both isolates, from the description of the colonial and microscopic morphology, including images of both isolates and a solid phylogenetic reconstruction. In addition, the region that was sequenced is not related to 16S rRNA, this was an oversight, and the sequence corresponds to the intergenic region associated with 18S and it is clearly seen that at the end of the sequence there is an erroneous gap, and this could be corroborated through a rigorous phylogenetic analysis using sequences from well-characterized type strains. In addition, the percentage of similarity obtained with this analysis must be provided. Because spp. for Talaromyces pinophilus?

 

In the section "Degradation kinetics study" there was a lack of including a control that corresponds to the experiments with mineral medium added with fluoranthene plus biomass inactivated by heat, this control is vital to evaluate the adsorption process of fluoranthene to fungal hyphae. Therefore, you are surely overestimating the degradation of fluoranthene by both fungal isolates. In fact, to know exactly the real percentage of degradation, the value corresponding to the inactivated biomass must be subtracted from the value of degradation from live cultures at each point of the kinetics. Regarding Figure 1, there are several inconsistencies between what is mentioned in the text and what is shown in Figure 1. For example, “803 mg/L was recorded for TpFLU12 at 8 d after the incubation”, and curiously on the Y axis of the mentioned graph it only reaches 720 mg/L (Fig. 1B), the same occurs in Fig. 1D. In addition, Figure 1, graphs must be homogenized, that the Y axes must be aligned in the three graphs, the size and type of font in both axes must be homogeneous in the 3 graphs.

 

In the section "Ligninolytic activities in the presence of fluoranthene", throughout this paragraph they report "optimal" enzymatic activities, however the results they show do not correspond to them, for this purpose different conditions should be evaluated such as pH, temperature, O₂ concentration, different fluoranthene concentrations, etc. To determine the optimal conditions for each enzyme evaluation, if the authors mean “maximum enzyme activities” they should modify “optimal” to “maximum”. In this sense, correct the text the maximum value reached for the activity of the laccase that was at 4 d. (Fig. 3A). The control of this experiment is not adequate since it does not have a source of C so that the fungi can grow, the equivalent of acetone (which is the solvent in which fluoranthene is dissolved) or glucose (universal source of C) had to be added. In Figure 3, graphics, size, and type of letters must be homogenized on both axes.

 

Why the intracellular enzymatic activity or of some enzyme of the cytoplasmic membrane was not evaluated?

 

In the discussion section, improve the writing when discussing the models to which the degradation kinetics of both strains are adjusted, mention everything corresponding to one strain and then to the other. Delete the last paragraph of the discussion, which is part of the guide for MDPI authors.

 

Materiales y métodos

4.4 Degradation of fluoranthene:

Change the term “fortified” to “BSM added with fluoranthene”

Please include what kind of plates you started from for the addition of the discs with mycelium, were they plates of basal medium added with fluoranthene or some plate containing some rich medium, which one?

 

“The initial concentration of 400 mg/L was used based on its zero fungal growth inhibition on both selected strains (data not shown)”.

What does that paragraph refer to?

Does the concentration of 400 mg/L correspond to the minimum inhibitory concentration (MIC) or what?

Adding the data of the other concentrations tested and what was the parameter to select the concentration of 400 mg/L, a table can be included as supplementary material.

 

4.5

Y se consideró el fluoranteno adsorbido a las paredes de los hongos para la evaluación de la degradación de cada punto de la cinética de degradación?

 

4.9

Clarify how the enzymatic extract is obtained, is it given any treatment before the enzymatic evaluations?

 

 

Minor comments:

 

Remove unnecessary capitalization of those words that are not at the beginning of the sentence throughout the entire manuscript, examples: Hexane, Azino, Nitrolotriacetic, Hydrogen, Ketoadipate, Benzo(b)fluoranthene, Benzene, Fluoranthene, Transformed, Manganese, Lignin, among others.

 

In the abstract: Line 22, include italics in “Tp”FLU12

In the Introduction: Line 80, change “Conversely” to “Besides”.

Mentioning that both isolates belong to the phylum Ascomycota, in the text it is only clear for Trichoderma but not for Talaromyces.

 

In Figure 4, include the asterisks on the line of significant differences for graph A.

Author Response

Response to Reviewer’s comments

Response to reviewers’ comments on Ms. Ref. No.: Catalysts-2279360, titled “Metabolic Biodegradation Pathway of fluoranthene by indigenous Trichoderma lixii and Talaromyces pinophilus spp.”

 

Dear Editor.

 

Catalysts,

 

The authors would like to thank the reviewers for their suggestions and consideration of our revised manuscript for publication in your reputable journal, Catalysts. We have revised the manuscript extensively throughout and outlined below the point-by-point responses to reviewers’ comments. The changes suggested by reviewers and other changes from the authors, in the revised manuscript are highlighted yellow where required.

 

Reviewer 1:

Major comments

 

1. The identification of the strains requires a thorough analysis to confirm if the strains correspond to the fungal genera mentioned in the manuscript. This is relevant since both fungal strains are mentioned from the title of the paper, it is only slightly commented (a single line) in the materials and methods section, but it leaves doubts because the accession number is the same for both strains. In addition, making a simple blast of the mentioned sequence (MK208694) displays other fungal genera. Therefore, it is necessary to include a more solid characterization of both isolates, from the description of the colonial and microscopic morphology, including images of both isolates and a solid phylogenetic reconstruction. In addition, the region that was sequenced is not related to 16S rRNA, this was an oversight, and the sequence corresponds to the intergenic region associated with 18S and it is clearly seen that at the end of the sequence there is an erroneous gap, and this could be corroborated through a rigorous phylogenetic analysis using sequences from well-characterized type strains. In addition, the percentage of similarity obtained with this analysis must be provided. Because spp. for Talaromyces pinophilus?

 

Author’s Response: The authors thank the reviewer for the comments which will be improving the quality of this manuscript. Kindly note a section 2.1 (page 3, line 110-134) has been added in revised manuscript. The section describes the through identification and characterization of both the strains. The correct submitted accession numbers for Trichoderma lixii strain FLU1 (MK208694) and for Talaromyces pinophilus strain FLU12 (MK208704) has been mentioned in “line 122 and 129”, respectively. Also, the characterization of the strains was based on molecular identification, and the protocol has been added as section 4.3 (lines “507 – 535”. The phylogenetic typing for the strains has been constructed and shown as Fig. S3. The percent of similarity obtained (99%) is mentioned in section 2.1, line 133-134. The authors apologize for wrongly mentioned 16S rRNA, and corrected as 18S rRNA, in section 4.3, line 515-516.

 

2. In the section "Degradation kinetics study" there was a lack of including a control that corresponds to the experiments with mineral medium added with fluoranthene plus biomass inactivated by heat, this control is vital to evaluate the adsorption process of fluoranthene to fungal hyphae. Therefore, you are surely overestimating the degradation of fluoranthene by both fungal isolates. In fact, to know exactly the real percentage of degradation, the value corresponding to the inactivated biomass must be subtracted from the value of degradation from live cultures at each point of the kinetics. Regarding Figure 1, there are several inconsistencies between what is mentioned in the text and what is shown in Figure 1. For example, “803 mg/L was recorded for TpFLU12 at 8 d after the incubation”, and curiously on the Y axis of the mentioned graph it only reaches 720 mg/L (Fig. 1B), the same occurs in Fig. 1D. In addition, Figure 1, graphs must be homogenized, that the Y axes must be aligned in the three graphs, the size and type of font in both axes must be homogeneous in the 3 graphs.

Author’s Response: The authors thank the reviewer for the comments but kindly note that to avoid over-estimation and under-estimation of the residual fluoranthene concentration, the manuscript has considered these problems and mitigated them following the processes below:

1. Recovery analysis was conducted and reported in “lines 575 -579” with an average fluoranthene recovery of 97.9 ± 7.17 %. This result implies that, analytically, there is no over-estimation/under-estimation of the residual fluoranthene concentration in the study.
2. To avoid over-estimation/under-estimation of the residual fluoranthene concentration, which might occur through fluoranthene biosorption by the fungi mycelia pellets. The residual fluoranthene concentration that might have been adsorbed to the fungi mycelia surface was extracted through the washing of fungi mycelia pellet with ethyl acetate (1:10 w/v) 5 times. This is mentioned in section 4.5, lines 557-560.
3. According to the study design's scope, the fungi's efficiency in degrading fluoranthene was monitored through the experimental set-up where a control sample (BSM + Fluoranthene + no fungus inoculation) and an experimental sample (BSM + Fluoranthene + fungus inoculation) were taken (section 4.4). Although. from literatures read before designing the study, no control (BSM + PAH compound + no fungi inoculation) has been reported with an overestimation of PAHs degradation. Below are some of the few peer-review journals with their experimental control design.

Agrawal, N., Verma, P. and Shahi, S.K. 2018. Degradation of polycyclic aromatic hydrocarbons (phenanthrene and pyrene) by the ligninolytic fungi Ganoderma lucidum isolated from the hardwood stump, Bioresour Bioprocess. 5 :11.

Experimental design

“To investigate the phenanthrene and pyrene degradation, mineral salt broth containing 20 mg/L phenanthrene and pyrene was taken separately. Then, 8 mm diameter, one culture mycelium disc was transferred in the broth media, control set-up containing mineral salt broth with phenanthrene and pyrene separately. Incubate the culture at 27 °C in a rotatory shaker incubator for 2, 5, 10, 15, 20, and 30 days”.

Agrawal, N., Shahi, S.K., 2017. Degradation of polycyclic aromatic hydrocarbon (pyrene) using novel fungal strain Coriolopsis byrsina strain APC5. Int Biodeterior Biodegradation 122, 69-81

Experimental design

“To investigate the pyrene degradation one fungal mycelium disc of 8 mm diameter was transferred in 20 mL mineral salt broth containing 20 mg/L pyrene, and incubated at 27 ˚C in a rotary shaker for 2, 4, 6, 8, 10, 12, 14, 16 and 18 days. Pyrene degradation and ligninolytic enzyme assay were examined at 2 days intervals. The control set-up contains mineral salt broth with pyrene under the same conditions as the experimental set”.

Bankole, P.O., Semple, K.T., Jeon, B.H., Govindwar, S.P., 2021. Biodegradation of fluorene by the newly isolated marine-derived fungus, Mucor irregularis strain bpo1 using response surface methodology. Ecotoxicol Environ Saf 208, 111619-111619

Experimental design

“The degradation experiments were carried out in 500 mL- Erlenmeyer flasks containing 100 mL mineral salt medium (MSM). The media was supplemented with different concentrations (100–500 mg L1) of fluorene, mycelia dry weight (0.2–2.0 g). In addition, the incubation temperature was varied between (25–40 ◦C) while the pH of the medium was varied between 5 and 9 with the addition of HCl and NaOH to the mixture. Abiotic control was made of mixture with no fungus mycelia. The experimental set-up were done in triplicates and kept for 5 days. Cultures were later filtered with Whatman filter paper to remove the solid and extract the fluorene in the mixture. After the experiment, the resultant fluorene in the mixture was extracted with ethyl acetate in a ratio 30:70. The filtrate was thereafter dried overnight with Na₂SO₄. The absorbance of the filtrates (experimental and control) containing suspended solids was taken with HPLC machine (Shimadzu, Japan) at a wavelength (λmax=263 nm) (Bressler et al., 2000)”.

Bankole, P.O., Semple, K.T., Jeon, B.H., Govindwar, S.P., 2020. Enhanced enzymatic removal of anthracene by the mangrove soil-derived fungus Aspergillus sydowii BPOI. Front Environ Sci Eng 14, 113

 

Experimental design

“The procedure was repeated for concentrations (200–500 mg/L) to obtain full calibration of the anthracene curve. Different anthracene concentrations were added to 30 mL of mineral salt media (MSM) in 250 mL containing A.sydowii mycelia (2 g). The setup was prepared in triplicates and kept for 72 h with flasks containing no fungus used as biocontrol (Ting et al., 2011).”

 

Álvarez-Barragán, J.,  Cravo-Laureau, C.,  Wick, L.Y. and Duran, R. (2021). Fungi in PAH-contaminated marine sediments: Cultivable diversity and tolerance capacity towards PAH, Mar Pollut Bull. 164 (2021), 112082.

 

Experimental design

“Between 80 and 100 mg of mycelia and conidia were recovered of MDAsw plates and inoculated in 80 mL flasks with 30 mL of MDsw (1% MD). Fluoranthene, phenanthrene, pyrene and benzo[a]pyrene were added from a stock solution prepared in acetone containing 20 mg/L of each hydrocarbon. Samples were set for 1 h before incubation to let acetone evaporate. An un-inoculated flask was used as abiotic control and PAHs concentration reference. Cultures were incubated in darkness for 20 days at 20 °C with gentle shaking at 80 rpm, to maintain culture conditions closer to that observed in the environment. Hydrocarbons were extracted after incubation by adding 30 mL of ethyl acetate and shacked for 15 min at 600 rpm. The recovery yield was estimated to be about 98% of the initial concentration using the abiotic controls as a reference”.

 

Regarding Figure 1, the inconsistency (803 mg/L was recorded for TpFLU12 at 8 d after the incubation) has been revised as “564.4 mg/L (line 138) was recorded for TpFLU12 at 16 d after the incubation period”. Also, “Total protein content of about 720 μg/mL at 10 d and 790 μg/mL at 12 d was recorded for TlFLU1 and TpFLU12, respectively” has been corrected to “protein content of about 643.4 μg/mL at 16 d and 566.5 μg/mL at 14 d was recorded for TlFLU1 and TpFLU12, respectively (lines 143-145)”

The axis in Fig 1A-D are aligned and font size and type are homogenized.

 

3:  In the section "Ligninolytic activities in the presence of fluoranthene", throughout this paragraph they report "optimal" enzymatic activities, however the results they show do not correspond to them, for this purpose different conditions should be evaluated such as pH, temperature, O₂ concentration, different fluoranthene concentrations, etc. To determine the optimal conditions for each enzyme evaluation, if the authors mean “maximum enzyme activities” they should modify “optimal” to “maximum”. In this sense, correct the text the maximum value reached for the activity of the laccase that was at 4 d. (Fig. 3A). The control of this experiment is not adequate since it does not have a source of C so that the fungi can grow, the equivalent of acetone (which is the solvent in which fluoranthene is dissolved) or glucose (universal source of C) had to be added. In Figure 3, graphics, size, and type of letters must be homogenized on both axes.

 

Author’s Response: "optimal enzymatic activities” has been revised to "maximum enzymatic activities in section 2.6 and all over the manuscript where required” lines 259, 261, 264, 266. Also, the control experiment is sufficient for this study’s claim because, according to the literature reviews, extracellular enzymes (ligninolytic enzymes) are are produced by fungi in response to the presence of PAHs (Álvarez-Barragán et al., 2021; Agrawal et al., 2017). The result showed that no ligninolytic enzyme activity was observed in control (BSM + fungi) due to the absence of the carbon source (Bankole et al., 2021). Thus, these findings suggest that the control experiment is sufficient in demonstrating that ligninolytic enzyme activity depends on fluoranthene's presence. Moreover, ligninolytic enzyme activities observed in the flask inoculated with each fungus provide evidence that these enzymes are only produced in the presence of fluoranthene. This observation has been reported in the below references.       

Bankole, P.O., Semple, K.T., Jeon, B.H., Govindwar, S.P., 2021. Biodegradation of fluorene by the newly isolated marine-derived fungus, Mucor irregularis strain bpo1 using response surface methodology. Ecotoxicol Environ Saf 208, 111619-111619

Agrawal, N., Shahi, S.K., 2017. Degradation of polycyclic aromatic hydrocarbon (pyrene) using novel fungal strain Coriolopsis byrsina strain APC5. Int Biodeterior Biodegradation 122, 69-81

Agrawal, N., Verma, P. and Shahi, S.K. Degradation of polycyclic aromatic hydrocarbons (phenanthrene and pyrene) by the ligninolytic fungi Ganoderma lucidum isolated from the hardwood stump, Bioresour Bioprocess. 5 (2018) 11.

 

Reviewer: In Figure 3, graphics, size, and type of letters must be homogenized on both axes.

Author’s Response:  Figure 3 has been revised.

 

Reviewer: Why the intracellular enzymatic activity or of some enzyme of the cytoplasmic membrane was not evaluated?

Author’s Response:  Preliminary study viz tolerance test (result not shown) using solid plate method reveal that the fungi could produce only extracellular enzyme in the presence of PAHs (low molecular weight and high molecular weight). Also, in the first 8 days of the degradation assay, intracellular enzymatic activities were not detectable. The para is mentioned in discussion section, Lines 453-457.

 

Reviewer: In the discussion section, improve the writing when discussing the models to which the degradation kinetics of both strains are adjusted, mention everything corresponding to one strain and then to the other. Delete the last paragraph of the discussion, which is part of the guide for MDPI authors.

Author’s Response: We would like to maintain the status quo in discussing each degradation kinetics model with the two fungi strains because it gives readers more clarity and better understanding than writing on each fungus with degradation kinetics models, as suggested. Also, “part of the guide for MDPI authors” has been expunged as advised.

 

Reviewer: 4.4 Degradation of fluoranthene:

Change the term “fortified” to “BSM added with fluoranthene”

Author’s Response: “fortified” has been changed to “BSM broth added with fluoranthene” in line 539.

Reviewer: Please include what kind of plates you started from for the addition of the discs with mycelium, were they plates of basal medium added with fluoranthene or some plate containing some rich medium, which one?

Author’s Response: “potato dextrose agar plate” has been used and mentioned in line 541.

 

Reviewer: “The initial concentration of 400 mg/L was used based on its zero fungal growth inhibition on both selected strains (data not shown)”. What does that paragraph refer to?

Author’s Response: It was juxtaposing the main reason for choosing 400 mg/L as our preferred concentration for the fluoranthene degradation study owing to prior results obtained during the tolerance test.

Reviewer: Does the concentration of 400 mg/L correspond to the minimum inhibitory concentration (MIC) or what?

Author’s Response: The fluoranthene concentration of 400 mg/L was the highest concentration for both fungi, which shows zero fungi growth inhibition due to the prior tolerance test assay.

Reviewer: Adding the data of the other concentrations tested and what was the parameter to select the concentration of 400 mg/L, a table can be included as supplementary material.

Author’s Response: This has been added to supplementary material as suggested as Table S2, section 4.4, line 547.

 

Reviewer: 4.5. And was fluoranthene adsorbed to the walls of fungi considered for the evaluation of the degradation of each point of the degradation kinetics?

Author’s Response: Yes, the residual fluoranthene concentration that might have been adsorped to the fungi mycelia surface was extracted through the washing of fungi mycelia pellet with ethyl acetate (1:10 w/v) 5 times before adding to residual fluoranthene extracted from the biomass-free degradation sample before analytical quantification of the residual fluoranthene as stated in lines “557 – 560”

 

Reviewer: 4.9. Clarify how the enzymatic extract is obtained, is it given any treatment before the enzymatic evaluations?

Author’s Response: Since we assay for the extracellular enzymes, the biomass-free degradation medium was used as the enzymatic extract.

 

Minor comments:

 

Reviewer: Remove unnecessary capitalization of those words that are not at the beginning of the sentence throughout the entire manuscript, examples: Hexane, Azino, Nitrolotriacetic, Hydrogen, Ketoadipate, Benzo(b)fluoranthene, Benzene, Fluoranthene, Transformed, Manganese, Lignin, among others.

Author’s Response: This has been affected as advised. e.g. Section 4.1

Reviewer: In the abstract: Line 22, include italics in “Tp”FLU12

Author’s Response: “TpFLU12” has been revised to “TpFLU12” line 22.

Reviewer: In the Introduction: Line 80, change “Conversely” to “Besides”.

Author’s Response: “Conversely” has been changed to “Besides” as advised in line 88.

 

Reviewer: Mentioning that both isolates belong to the phylum Ascomycota, in the text it is only clear for Trichoderma but not for Talaromyces.

Author’s Response: Kindly note both Trichoderma and Talaromyces belong to the phylum Ascomycota.

 Reviewer: In Figure 4, include the asterisks on the line of significant differences for graph A.

Author’s Response:*** has been added, See Fig 4.

Reviewer 2 Report

Bio-catalytic degradation of fluoranthene has been studied. The subject is interesting and valuable. However, there are some points which should be addressed and discussed in the revised version, before the final publication. I suggest revision of the manuscript based on the following points:

 

1.       It has been mentioned that “Among the ubiquitous group of organic xenobiotics found in the various matrix of the ecosystem, polycyclic aromatic hydrocarbons (PAHs) have become a major environmental priority owing to their carcinogenicity, genotoxicity, cytotoxicity and mutagenic effects, which are characterized by low aqueous solubility and high lipophilicity to biological functions”. This should be improved and supported as follows: “Among the ubiquitous group of organic xenobiotics found in the various matrix of the ecosystem, polycyclic aromatic hydrocarbons (PAHs) have become a major environmental priority owing to their carcinogenicity [Carcinogenicity of polycyclic aromatic hydrocarbons], genotoxicity [Size-dependent genotoxicity of graphene nanoplatelets in human stem cells], cytotoxicity [Toxicity of Graphene and Graphene Oxide Nanowalls Against Bacteria] and mutagenic effects [Atmospheric Environment Volume 60, December 2012, Pages 375-382], which are characterized by low aqueous solubility and high lipophilicity to biological functions”.

 

2.       In Figure 2, the products need to be confirmed either by suitable characterizations such as CGMS and FTIR or supported by suitable references.

 

3.       It has been mentioned that “Apart from its carcinogenicity, mutagenicity, and teratogenicity, preclinical studies have shown a direct link between PAH exposure, oxidative stress, CVD illness and mortality development”. This can be completed by mentioning the endocrine disruption effect due to PAH exposure. Reference in this field could be included.

 

4.       The data points in Figure 4 need error bar. The same for Figures 3A and B.

 

5.       Some of the PAH can act as carbocatalysts in degradation of hazardous aromatic molecules. Papers published recently on this side could be cited. This should be addressed in the revised version.

 

6.       The efficiency of the degradation should be evaluated and then compared with the related published works.

 

7.       Could the authors comment about the dependence of the efficiency on the initial concentration of PAH? One guess is that at high concentrations, the efficiency decreases significantly. Please discuss using suitable supports.       

Author Response

Response to Reviewer’s comments

Response to reviewers’ comments on Ms. Ref. No.: Catalysts-2279360, titled “Metabolic Biodegradation Pathway of fluoranthene by indigenous Trichoderma lixii and Talaromyces pinophilus spp.”

 

Dear Editor.

 

Catalysts,

 

The authors would like to thank the reviewers for their suggestions and consideration of our revised manuscript for publication in your reputable journal, Catalysts. We have revised the manuscript extensively throughout and outlined below the point-by-point responses to reviewers’ comments. The changes suggested by reviewers and other changes from the authors, in the revised manuscript are highlighted yellow where required.

 

Reviewer 2:

Major comments

Reviewer comments 1.  It has been mentioned that “Among the ubiquitous group of organic xenobiotics found in the various matrix of the ecosystem, polycyclic aromatic hydrocarbons (PAHs) have become a major environmental priority owing to their carcinogenicity, genotoxicity, cytotoxicity and mutagenic effects, which are characterized by low aqueous solubility and high lipophilicity to biological functions”. This should be improved and supported as follows: “Among the ubiquitous group of organic xenobiotics found in the various matrix of the ecosystem, polycyclic aromatic hydrocarbons (PAHs) have become a major environmental priority owing to their carcinogenicity [Carcinogenicity of polycyclic aromatic hydrocarbons], genotoxicity [Size-dependent genotoxicity of graphene nanoplatelets in human stem cells], cytotoxicity [Toxicity of Graphene and Graphene Oxide Nanowalls Against Bacteria] and mutagenic effects [Atmospheric Environment Volume 60, December 2012, Pages 375-382], which are characterized by low aqueous solubility and high lipophilicity to biological functions”.

Author’s Response:  The authors thank the reviewer for the comment which will be improving the quality of the manuscript. As suggested by the reviewer,  “Among the ubiquitous group of organic xenobiotics found in the various matrix of the ecosystem, polycyclic aromatic hydrocarbons (PAHs) have become a major environmental priority owing to their carcinogenicity, genotoxicity, cytotoxicity and mutagenic effects, which are characterized by low aqueous solubility and high lipophilicity to biological functions” is revised as: “Among the ubiquitous group of organic xenobiotics found in the various matrix of the ecosystem, polycyclic aromatic hydrocarbons have become a major environmental priority owing to the carcinogenicity, size-dependent genotoxicity of graphene nanoplatelets in human stem cells, toxicity of graphene and graphene oxide nano-walls against bacteria and mutagenic effects”. Lines 29-33.

Reviewer comments 2.       In Figure 2, the products need to be confirmed either by suitable characterizations such as CGMS and FTIR or supported by suitable references.

Author’s Response: Section 4.7 and 4.8 describe the GCMS and FTIR analysis of the degradation products. Figure 2 caption has been revised as “The proposed fluoranthene metabolic pathway by TlFLU-1 and TpFLU-12. The un-bold arrows denote an unknown transient product which were not detected by GCMS analysis” in lines 224 - 225. Also, section 4.7 showed how the products mapped out in the pathways were identified. Thus, the obtained GCMS spectral and FTIR are in the supplementary material Table S1. This method has been employed by different authors in the references below when characterizing the degradation products.

Agrawal, N., Shahi, S.K., 2017. Degradation of polycyclic aromatic hydrocarbon (pyrene) using novel fungal strain Coriolopsis byrsina strain APC5. Int Biodeterior Biodegradation 122, 69-81

Methods used: GCMS and FTIR

Agrawal, N., Verma, P. and Shahi, S.K. 2018. Degradation of polycyclic aromatic hydrocarbons (phenanthrene and pyrene) by the ligninolytic fungi Ganoderma lucidum isolated from the hardwood stump, Bioresour Bioprocess. 5 :11.

Methods used: GCMS and FTIR

Bankole, P.O., Semple, K.T., Jeon, B.H., Govindwar, S.P., 2021. Biodegradation of fluorene by the newly isolated marine-derived fungus, Mucor irregularis strain bpo1 using response surface methodology. Ecotoxicol Environ Saf 208, 111619-111619

Methods used: GCMS and FTIR

Bankole, P.O., Semple, K.T., Jeon, B.H., Govindwar, S.P., 2020. Enhanced enzymatic removal of anthracene by the mangrove soil-derived fungus Aspergillus sydowii BPOI. Front Environ Sci Eng 14, 113

Methods used: GCMS and FTIR

Reviewer comments 3. It has been mentioned that “Apart from its carcinogenicity, mutagenicity, and teratogenicity, preclinical studies have shown a direct link between PAH exposure, oxidative stress, CVD illness and mortality development”. This can be completed by mentioning the endocrine disruption effect due to PAH exposure. Reference in this field could be included.

Author’s Response: As suggested, “Apart from its carcinogenicity, mutagenicity, and teratogenicity, preclinical studies have shown a direct link between PAH exposure, oxidative stress, CVD illness, mortality development” is revised as, “Apart from its carcinogenicity, mutagenicity, and teratogenicity, preclinical studies have shown a direct link between PAH exposure, oxidative stress, CVD illness, mortality development and endocrine disruption” lines 55-57.  Ref “Kubincová, P., Sychrová, E., Raška, J., Basu, A., Yawer, A., Dydowiczová, A., Babica, P. and Sovadinová, I. “Polycyclic aromatic hydrocarbons and endocrine disruption: Role of testicular gap junctional intercellular communication and connexins”. Toxicological sciences, 169, no. 1 (2019):70-83.” Is cited.

Reviewer comments 4. The data points in Figure 4 need error bar. The same for Figures 3A and B.

Author’s Response: The error bars on the figures mentioned has been added but are very low and not really showing.

Reviewer comments 5. Some of the PAH can act as carbocatalysts in degradation of hazardous aromatic molecules. Papers published recently on this side could be cited. This should be addressed in the revised version.

Author’s Response: As suggested, a paragraph, “Some PAHs exhibit carbocatalytic activity as they can act as catalysts to break down hazardous aromatic molecules due to their unique electronic structure and reactivity (Hung et al., 2022). PAHs have multiple fused aromatic rings with pi-electrons that make them highly reactive and able to act as electron donors or acceptors when exposed to hazardous aromatic molecules, they undergo redox reactions, breaking them into less toxic compounds (Kumari and Lakhani, 2018).” Has been added in introduction section, lines 40-45.

Reviewer comments 6. The efficiency of the degradation should be evaluated and then compared with the related published works.

Author’s Response: The biodegradation efficiency of the fungal strains used in our study has been discussed in lines 354 – 360 as “The increment in fluoranthene degradation rate by the fungi with an increase in incubation period is comparable to other reports where PAH compounds degradation is proportional to the incubation period [23, 33]. Similarly, the ability of the tested fungi to exhibit higher and faster fluoranthene degradation (99 %) at 12 d of incubation period compared to earlier reported white-rot fungus (Pleurotus eryngii) where 80% of fluoranthene was degraded at 30 d after incubation period under agitated condition [27]”.

Reviewer comments 7.   Could the authors comment about the dependence of the efficiency on the initial concentration of PAH? One guess is that at high concentrations, the efficiency decreases significantly. Please discuss using suitable supports. 

Author’s Response: The comment has been discussed in discussion section, “The efficiency of PAHs degradation can decrease at higher concentrations, possibly due to mass transfer limitations or adsorbent material saturation. Other factors such as pH, temperature, and the type and concentration of enzymes, can also affect the efficiency of PAH removal. For example, during the biodegradation of anthracene by Aspergillus sydowii strain bpo1, high concentration of anthracene, extracellular enzymes activities (laccase, lignin and Manganese peroxidase), pH and temperature individually have significant impact on the biodegradation efficiency (Bankole et al., 2020), lines 360-366.

Bankole, P.O., Semple, K.T., Jeon, B.H., Govindwar, S.P., 2020. Enhanced enzymatic removal of anthracene by the mangrove soil-derived fungus Aspergillus sydowii BPOI. Front Environ Sci Eng 14, 113

 

 

Round 2

Reviewer 1 Report

The authors considered several suggestions, and the paper was improved in several sections. However, the section on the identification of the two isolates is still not properly addressed. This is the point that continues to be of concern since, as I mentioned from the first review, it is very important because the names of the strains are included from the title of the work, and perhaps a good phylogenetic analysis will yield different results. Although an effort was made to add a section on molecular analysis to the methods section, there are several inconsistencies in that analysis. Firstly, the authors did not sequence the 18 rRNA gene, but rather the "intergenic ITS1-5.8S-ITS2 region of the ribosomal RNA (rRNA)" region, as the authors themselves described and submitted to GenBank, this must be corrected in the manuscript, particularly in the materials and methods section, and in the results section. The analysis of the sequences included in this new version of the manuscript is very basic. In fact, it is logical that the sequences of the strains in this study show 100% similarity with the same sequences that the same authors uploaded to GenBank, this was expected.

To take an example, the BLAST displays similarity with: an isolate of Penicillium, with Chaetomium and later with isolates of Talamyces muroii, Talomyces sp., Talomyces pinophilus, among others (attached screenshot of the blast with the MK208694 sequence). This approach is very basic, and a phylogenetic analysis must be carried out that includes the sequences of the type strains of the genus Talomyces or the genus that is most closely related. The programs that were included in the methodology are sufficient to carry out this analysis, so it would be appreciated if they were applied and that these results were shown in the corresponding section. The phylogenetic reconstructions (phylogenetic trees for each strain) with the type sequences of other species related to each genus, will yield a % similarity in MEGA, that is the % that the authors should include in the manuscript, not the one that yields a quick analysis of BLAST. In addition, it would be necessary to include the taxonomic criteria that would be used to assign the isolates to a certain genus and/or fungal species.

 

 

 

 

 

In addition, the colony morphology portion of the description should include which culture medium was used in the determination, colony morphology (aerial mycelium color, vegetative mycelium color, whether there is diffusible pigmentation, radial or irregular growth), and microscopic morphology [type of mycelium (coenocytic, septate), shape of the conidium, type of spores, coloration of spores, bifurcations of the hyphae]. Usually the microscopic description is carried out by growth in a microculture. In fact, it would be appreciated if you present an integrated figure of the colonial and microscopic morphology of each isolate, instead of figures S1 and S2.

 

For this, I suggest that the authors review some strain identification works and take inspiration from them, such as:

 

doi.org/10.3389/fmicb.2022.985874

doi.org/10.3390/jof7110993

doi.org/10.1007/s11033-021-06321-0

doi.org/10.1007/s12010-014-1336-x

 

 

Minors:

Unnecessary capital letters continue to appear, such as Benzo(b)fluoranthene on line 11; Ketoadipate, line 25; Lignino-, line 88; Manganese, line 365, among others. Revise the entire manuscript and lowercase words that do not require capitalization.

Comments for author File: Comments.pdf

Author Response

The authors thank the reviewer’s comments that will improve the manuscript quality. Although we had attended many points from reviewer in first revision, we missed some important points to attend. The microscopic analysis of the strains were performed and new phylogenetic tree were constructed. Please see below the response to the reviewer’s comments and we hope the reviewer will agree with the revision.

 

1. The phylogenetic tree has been reconstructed for each fungal strain as shown in fig. s3a and fig. S3b and replaced with Figure S3. Also, the phylogenetic tree interpretation has been added to the result section in lines 141 – 145. Also see methods section lines 534-537.
2. 18S rRNA gene has been revised to “intergenic ITS1-5.8S-ITS2 region of the ribosomal DNA (rDNA)” in lines 518 – 519. Also, 18S rRNA has been omitted from lines 111, 121 and 128.
3. The taxonomic criteria were used to assign the isolates to a certain genus and/or fungal species based on their molecular identification with their percentage (100 %) identity and the accession number obtained from NCBI after the nucleotide sequence had been deposited early with MK208694 for Trichoderma lixii and MK208704 for Talaromyces pinophilus
4. A detailed description of the fungi colony morphology on the culture medium (PDA plate) which entails the aerial mycelium colour, vegetative mycelium colour, and diffusible pigmentation used, has already been added in the first revision in lines 111 - 127.
5. A microscopic morphology that entails type of mycelium, its radial or irregular growth, the shape of the conidium, type of spores, the colouration of spores, and bifurcations of the hyphae are now added to lines 121 -124 for Trichoderma lixii (Fig. S1B) and lines 131 -139 for Talaromyces pinophilus (Fig.S2B).
6. The taxonomic criteria shown in fig. S3A and S3B were used to assign the isolates to a certain genus and/or fungal species. Please also see line 125-126 and 137-138.

Reviewer 2 Report

The revisions are acceptable. 

Author Response

The authors are thankful to the reviewer for accepting the revision.