Trichoderma: Evaluation of Its Degrading Abilities for the Bioremediation of Hydrocarbon Complex Mixtures

Round 1

Reviewer 1 Report

Line 113: the reference Martin & Rygiewicz 2005 is not reported in the bibliography

Line 142: is there any data between 0 and 45 days?

Line 160: is the % content an average value? 4 or 8 replicates?

Line 179: what is “Per” activity ? Peroxidase?

Line 252: move the sentence “U test …..” from fig 3

Line 257: Figure 4: what are the variables (in PC1 and PC2) used for the PCA? Why only for F26?

Line 241-245: according to Figure 3: T.h. F12 degradation of aromatic compounds is 5% (71-66) while aliphatic compounds increase is 4%. For T.a.F1020 aromatic compounds decrease is 4% corresponding to 4% of aliphatic compound increase. Is it possible that such a small difference (1%) changes the significance?

Line 247-251: it is not clear: the application of F26 reduces the aromatic compounds by 9% (74-65) while the aliphatic compounds increase by 7% (30-27) while the application of F58 reduces the aromatic compounds by 15% (73-58) and the aliphatic compounds increase by 14% (37-23). Therefore F58 seems to be more effective than F26.

Line 259: considering the aliphatic fraction C1-C20 + C20-C50, from figure 5 the increase of fraction seems to be about 2 percentage units while from figure 3 the increase is 7% percentage units. Are the reported data the average values of 4 samples? Moreover why data only for F26 sample?

Line 289-293: it looks more like an introduction rather than a discussion of experimental data

Line 303-306: the reported data have not been verified experimentally, moreover the same oil was used

Line 308-320: already mentioned elsewhere in the text

Line 328-320: already mentioned

Line 322-322: move in the introduction

Figure 5: why only data related to F26 sample?

Line 380: deepen knowledge regarding this topic

Line 396: not demonstrated with the reported data considering that authors use the same oil

Line 363-371: The reference 56 is missing from the text

Line 420 and 527: not compliant with the editorial standard

Author Response

Response to Reviewer 1 Comments

1. Line 113: the reference Martin & Rygiewicz 2005 is not reported in the bibliography

The reference has been inserted, and all the bibliography has been revised.

2. Line 142: is there any data between 0 and 45 days?

No, we only have data for 0 and 45 days. We decided to choose a time-lapse long enough to detect the change in the tests and to limit the costs.

3. Line 160: is the % content an average value? 4 or 8 replicates?

Yes, it is an average value of 4 replicates. Not to mislead the reader, we have removed the sentence at lines 141-142

4. Line 179: what is "Per" activity ? Peroxidase?

Yes, it is peroxidase. The abbreviation's meaning has been added at line 79, where the word appears the first time.

5. Line 252: move the sentence "U test ….." from fig 3

Done.

6.Line 257: Figure 4: what are the variables (in PC1 and PC2) used for the PCA? Why only for F26?

In Principal Components Analysis (PCA), PC1 and PC2 represent the two principal components that have been computed during this multivariate analysis. They depict the maximum variance present in the samples' data so that the PCA shows the samples in the graph distanced according to the difference among them. In this case, having axis PC1 78% of variance and axis PC2 14% of variance, our PCA shows 92% of the variance present in the samples (data regarding the hydrocarbon microclasses). This PCA confirms graphically the results of the PERMANOVA test performed on the modification of hydrocarbon microclasses by T. harzianum F26 between T0 and T45. In fact, the samples separate drastically in two groups according to the sampling time along the PC1 axis (the one that shows most variability). 

In order not to burden the text with many graphs we have first reported the graphs showing the activity of all the strains on the oil divided by macroclasses, and then we showed the graph of oil degradation divided into microclasses only of F26 Because it is the only statistically significant result. However, if it could be helpful, we will add the others in the article as well.

7. Line 241-245: according to Figure 3: T.h. F12 degradation of aromatic compounds is 5% (71-66) while aliphatic compounds increase is 4%. For T.a.F1020 aromatic compounds decrease is 4% corresponding to 4% of aliphatic compound increase. Is it possible that such a small difference (1%) changes the significance?

We have modified Figure 3 by transforming the graphical representation of the data from pie charts to bar charts. Thanks to your comment; we realized that bar charts are more explanatory of our data because we evaluated the oil degradation with percentages, but the hydrocarbon fraction of such a complex material does not represent the totality of the material itself. As you can see better in this new version of Figure 3, T. harzianum F12 increased the Aliphatics from 8.56% ± 1,00% to 10,84% ± 0.89%, while T. asperellum F1020 increased them from 9.34% ± 0,58% to 10,66% ± 0.59%. So in the case of T. harzianum F12, this increase was found statistically significant (Manova, p=0.002267, two-tailed (Wilcoxon) Mann-Whitney U test with Monte Carlo permutation p=0.0282), while this was not the case for T. asperellum F1020 (Manova, p=0.09293, two-tailed (Wilcoxon) Mann-Whitney U test with Monte Carlo permutation p=0.0549).

8. Line 247-251: it is not clear: the application of F26 reduces the aromatic compounds by 9% (74-65) while the aliphatic compounds increase by 7% (30-27) while the application of F58 reduces the aromatic compounds by 15% (73-58) and the aliphatic compounds increase by 14% (37-23). Therefore F58 seems to be more effective than F26.

We did not consider F58 as interesting as F26 because, as you can see from the new Figure 3, F58 statistically increases the aliphatic from 8.58% to 15.39%, but does not significantly reduce the aromatic fraction. F26 instead gives significant results for both aliphatic and aromatic compounds.

9. Line 259: considering the aliphatic fraction C1-C20 + C20-C50, from figure 5 the increase of fraction seems to be about 2 percentage units while from figure 3 the increase is 7% percentage units. Are the reported data the average values of 4 samples? Moreover why data only for F26 sample?

Figure 5: why only data related to F26 sample?

As explained thoroughly in point 7, we modified Figure 3 by transforming the graphical representation of the data from pie charts to bar charts. The discrepancies between the previous F26 pie chart and its bar graph (Fig. 4) were due to the differences in the construction of the two types of graphs. We then uniformed the graphs in order not to be misleading for the reader.

As we said in response to observation 6, we showed the graph of oil degradation divided into microclasses only of F26 because it is the only statistically significant result. However, if it could be helpful, we will add the others in the article as well.

10. Line 289-293: it looks more like an introduction rather than a discussion of experimental data

We decided to remove these statements.

11. Line 303-306: the reported data have not been verified experimentally, moreover the same oil was used

Line 308-310: already mentioned elsewhere in the text

Line 396: not demonstrated with the reported data considering that authors use the same oil

We agree with these comments and decided to remove all these statements.

12. Line 318-320: already mentioned

Line 380: deepen knowledge regarding this topic

All the paragraph has been revised

13. Line 322-322: move in the introduction

We think that these sentences (now from line 327) help to argue the result reported just before.

14. Line 363-371: The reference 56 is missing from the text

Reference 56 has been added (line 384).

Line 420 and 527: not compliant with the editorial standard

The bibliography has been checked and fixed based on the editorial standard.

Author Response File: Author Response.pdf

Reviewer 2 Report

The authors present and interesting study on the abilities of Trichoderma spp. to degrade used engine oil. The study is clearly presented and supported by the results. The minor comments and corrections are highlighted directly in the pdf file. 

I would suggest the authors, if they have the data, to give the quantitative measurements of Trichoderma growth on media supplemented with 1% engine oil instead of only visual evaluation (+, ++ or +++; Table 2)

Similarly, in Fig. 6, the authors could show comparison of a strain with high activity vs. low activity for all three tests, not only for guaiacol (B, C)

page 11 lines 337-338 - i would remove or reformulate the statement, that T. harzianum is the most suitable species for bioremediation - the authors did not confront a variety of species for their capacity of bioremediation, they do not have supporting results for this statement. 

 

Comments for author File: Comments.pdf

Author Response

Response to reviewer 2

1. I would suggest the authors, if they have the data, to give the quantitative measurements of Trichoderma growth on media supplemented with 1% engine oil instead of only visual evaluation (+, ++ or +++; Table 2)

We do not have quantitative data based on fungal biomass but we based on plate coverage percentage. We have added a more exhaustive explanation of this test (lines 133-140) and new description to Table 2.

2. Similarly, in Fig. 6, the authors could show comparison of a strain with high activity vs. low activity for all three tests, not only for guaiacol (B, C)

Fig. 6 has been updated following this suggestion.

3. page 11 lines 337-338 - i would remove or reformulate the statement, that T. harzianum is the most suitable species for bioremediation - the authors did not confront a variety of species for their capacity of bioremediation, they do not have supporting results for this statement.

The statement at line 337-338 has been removed.

The entire text has been corrected following the minor comments the reviewer made directly on the file.

 

Author Response File: Author Response.pdf

Reviewer 3 Report

The manuscript by Daccò et al. reports a study aiming to evaluate the ability to degrade hydrocarbons from engine oils of Trichoderma species.

Four strains of Trichoderma (three classified, by morphological and biomolecular analysis, as belonging to T. harzianum and one to T. asperellum) were inoculated with 1% of used engine oil and their grown capacity was evaluated as well as their ability to modify the composition of the oil (determined by GC-MS). At the same time, the possible mechanism of activity was investigated by detecting the Lignolytic Enzymatic Activity by different colorimetric assays. The results showed that the three strains of T. harzianum (and in particular F26 strain) were more tolerant to the oil medium and more able to modify the oil composition, probably because they were isolated from contaminated soils, being therefore potentially used as bioremediation agents.

In the reviewer’s opinion, the topic of the study can be of interests for the scientific community and the methodology applied is adequate. However, some points should be clarified before the publication of the manuscript:

1. The novelty of the present manuscript if compared to the literature studies on the crude oils degradation abilities of Trichoderma fungi (see ref. [26], [42, [43], ….) should be presented
2. Concerning the evaluation of the hydrocarbon degradation ability, some weakness can be observed; in particular, how was quantification carried out? By area percentage determination? At least an internal standard should be added because the authors are only measuring a percentage variation (if one class is decrease, another will increase) but it would be more interesting to see the absolute decrease of the analytes. Furthermore, by the identification point of view, the determination of retention indices (and their comparison with literature data) is mandatory for a correct determination of the identity of the compounds since several isomers are probably present
3. Considering the colorimetric screening, why the results were not repeated in presence of the oil? The comparison of the results would be interesting and the results more complete

Author Response

Response to reviewer 3 comments

1. The novelty of the present manuscript if compared to the literature studies on the crude oils degradation abilities of Trichoderma fungi (see ref. [26], [42, [43], ….) should be presented

The novelty of the manuscript has been presented in the discussion section (lines 307-315).

2. Concerning the evaluation of the hydrocarbon degradation ability, some weakness can be observed; in particular, how was quantification carried out? By area percentage determination? At least an internal standard should be added because the authors are only measuring a percentage variation (if one class is decrease, another will increase) but it would be more interesting to see the absolute decrease of the analytes.

The purpose of this work was a qualitative evaluation of the ability of Trichoderma strains to grow on used engine oil and how they can change the composition of the oil. In the future, we intend to carry out more in-depth analyses focusing on the classes of hydrocarbons that were the most attacked by F26 (the fungus that was found to be the most performing) and in this case to make a quantitative estimate, inserting a standard,  evaluating the absolute and not relative variations.

3. Furthermore, by the identification point of view, the determination of retention indices (and their comparison with literature data) is mandatory for a correct determination of the identity of the compounds since several isomers are probably present.

We have identified all the peaks we found by comparing them with databases, and we know that the identifications are often not certain. In these cases, to increase the certainty of identification, it is possible to do an additional GC/MS analysis by injecting a standard hydrocarbon mixture and then calculate the Kovats Retention Indices. In this work, we focused on distinguishing among hydrocarbon classes, and not between the various isomers. For this purpose, we grouped the hydrocarbons following the characteristic ions reported in the cited article [ Hostettler, F.D.; Lorenson, T.D.; Bekins B.A. Petroleum fingerprinting with organic marker, Environ. Forensics 2013 14(4), 262-277. doi: 10.1080/15275922.2013.8436111] and shown in Table 1S added in the supplementary materials. Therefore, the presence of isomers should not affect the composition of hydrocarbon classes, as any isomer would still fall within the same family.

4. Considering the colorimetric screening, why the results were not repeated in presence of the oil? The comparison of the results would be interesting and the results more complete.

For this test, we followed the mentioned protocols, but in preliminary work, we tried to perform the tests in Petri dishes with the addition of oil, but the colour of the oil itself made it impossible to appreciate the colorimetric change around the growing colony.

Author Response File: Author Response.pdf

Reviewer 4 Report

The article is relevant to the scope of the journal. The authors have evaluated four strains of Trichoderma as potential agents for bioremediation of hydrocarbon complex mixtures. This group has expertise in uncovering and characterizing fungal strains from extreme environments and this is a potentially impactful application to look into. The four strains were evaluated for their activity against engine oil and products were assayed using GC-MS, and these products were further identified and classified into different subgroups of hydrocarbon by products. As a possible explanation of their modes of actions, the authors further looked into enzymatic assays for laccase and peroxidase which proved inconclusive for all 4 strains.

Some other observations:

1. Few grammatical errors are highlighted in the manuscript with suggested corrections.
2. Table 2: Quantification seems arbitrary and warrants explanation for what the metrics mean in terms of the scale of growth represented
3. Materials and methods section are written in great detail and requires very little in terms of further inclusion.
4. Figure 2. Needs reformatting – the legend is sandwiching a line from the manuscript
5. I did not find any of the GC-MS data directly shown which is a concern for me. I would like to at least some of the representative GC-MS data in a supplemental section.

Comments for author File: Comments.pdf

Author Response

Response to reviewer 4 comments

1. Few grammatical errors are highlighted in the manuscript with suggested corrections.

The grammatical errors have been corrected based on the suggested corrections and in Fig. 1, the four strains have been highlighted.

2. Table 2: Quantification seems arbitrary and warrants explanation for what the metrics mean in terms of the scale of growth represented

We have added a more exhaustive explanation of this test (lines 133-140), and Table 2 has been updated, with a new description.

3. Figure 2. Needs reformatting – the legend is sandwiching a line from the manuscript

Figure 2 has been reformatted.

4. I did not find any of the GC-MS data directly shown which is a concern for me. I would like to at least some of the representative GC-MS data in a supplemental section.

Thanks to these precious suggestions, we have added the GC/MS data as supplementary materials. Figure 1S shows the chromatograms of one of the four replicates for each fungus at T0 and T45. The chromatograms have been divided into the three macroclasses indicated in the article (aliphatics, aromatics and other compounds). In the same way, the chromatograms in Figure 2S show the oil treated with F26 at T0 and T45, divided in microclasses.

Round 2

Reviewer 1 Report

in my opinion the revised paper can be accepted for publication in the Applied Sciences journal.

line 115 remove brackets

Reviewer 3 Report

Althoght the quantification method is, in the reviewer's opinion, questionable and at lest an internal standard should be added, the Authors satisfactorily replied to all the revier's comments and the manuscript is therefore acceptable for pubblication