Degradation of Polycyclic Aromatic Hydrocarbons by Co-Culture of Pleurotus ostreatus Florida and Azospirillum brasilense

Round 1

Reviewer 1 Report

 

 

The abstract clearly describes that the objective of the work is the degradation of PAH by the association of a fungus, known for this potential, and a bacterium.

Some suggestions:

1.       Introduction 

1.1   Briefly include contextualization of the relationship between enzymes and biodegradation processes.

1.2   Characterize the potential of the organisms studied, including that the bacterium can also produce the enzymes studied.

 

2.       Materials and Methods

2.1 Line 87. Include reference to the gravimetric method

2.2 Line 98. Standardize the forms of entry of values and quantities, this is different from the previous paragraphs

2.3 Line 100. The final total concentration of 5 mg per 100 ml of each PAH?

2.4 Line 101. How many microorganisms were added?

2.5 Line 119. What formula was used to arrive at U/ml? We have a serious problem with the incorrect use of formulas for this group of enzymes. We will only solve this problem by not omitting this information.

2.6 Has the adsorption of PAHs on the mycelium not been evaluated? We know that extraction with chloroform alone does not release material adsorbed by the hyphae. It is necessary to break the mycelium beforehand.

3.       Results

3.1 Line 126. Include the methodology in the previous part.

3.2 Line 139. I think this conclusion is hasty at this point, without discussion.

3.3 Line 143. As it seems that the mycelium did not break down, we cannot say that only the reduction in concentration is the result of degradation (figure 1), at this time of presentation of the results. After evaluating the metabolites, yes, we can say that there was degradation. However, not every % decrease in concentration can be the result of degradation. If the adsorption has not been evaluated, I suggest changing, at this point the use of the word degradation. Perhaps Disappearance like this in the picture.

3.4 Line 181. Reference?

3.5 Line 178. Make this experiment clearer in the methodology. Got confused

3.6 Line 223. This goes into the methodology

3.7 Line 239. This entire part of the IAA was not presented in the method.

As I understand it, there was induction with exogenous IAA and the evaluation of IAA as a metabolic of microorganisms. That's it? How were these experiments?

4.       Discussion

4.1 Line 250 to 269: This is the introduction, not a discussion.

4.2 Line 284. I believe it is not a valid reference. What is the difference between the cited work and the current work?

4.3 Line 304. It's the same information as the result. What's the discussion?

4.4 Line 311. Reference? These same retention times were found in other works in the literature. Even with other microorganisms? The discussion must go in that direction.

4.5 Line 317. “This may suggest the similar mechanisms in the initial attack of PAH molecules in the organisms studied.” Your work has no basis for this claim. What do the other works say? How is the metabolite pathway of each organism, and what are the intermediaries?

4.6 Line 321. Reference

4.7 Line 335. In this sentence and others, the scientific names are not in italics. Review.

4.8 Line 343. What is the IAA? Include in the introduction and emphasize the importance of the discussion

4.9 Is the addition of IAA interesting for a bioremediation process?4.10 Many studies have shown that in some cases the metabolites generated in the degradation process are more toxic. There are simple experiments that must be carried out by the group to really confirm the potential of the proposed study.

4.11 Correct the scientific names of the figures

4.12 The discussion should be further worked on. I highlight the lack of discussion of metabolites and their importance in the decontamination process.

 

 

Author Response

1. Briefly include contextualization of the relationship between enzymes and biodegradation processes.

As a promising option, fungal enzymes are regarded as a powerful choice for degradation of PAHs. Ligninolytic fungi have been extensively studied because they produce extracellular enzymes with extremely reduced substrate specificity. The ligninolytic system contains three peroxidase (lignin peroxidase, Mn-peroxidase, and versatile peroxidase) and laccase. The involvement of these enzymes to degradation of PAHs was shown many studies. The first attack of molecule of PAH resulted in formation of corresponding quinones. In addition to the mentioned enzymes other fungal enzymes, such as cytochrome P450 monooxygenase, epoxide hydrolase, and some dioxygenases can participate in PAH degradation [Kadri et al., 2016]. More often in bacterial degradation pathways, the PAHs is activated by incorporating molecular oxygen directly with the help of mono- or dioxygenases [Srivatstava, Kumar 2019]. More often in bacterial degradation pathways, the PAHs is activated by incorporating molecular oxygen directly with the help of mono- or dioxygenases [Srivatstava, Kumar 2019]. Usually, the first step in the aerobic bacterial degradation of PAHs is the hydroxylation of an aromatic ring via a dioxygenase, with formation of a cis-dihydrodiol, which gets rearomatized to a diol intermediate by the action of a dehydrogenase. These diol intermediate may then be cleaved by intradiol or extradiol ring-cleavage dioxygenases, leading to intermediates that can be involved to TCA cycle [Ghosal et al., 2016]. We added the information on relationship between enzymes and biodegradation process suggest.

 2. Characterize the potential of the organisms studied, including that the bacterium can also produce the enzymes studied.

Wood-inhabiting basidiomycetes of the genus Pleurotus belong to ligninolytic fungi, which are some of the most active degraders of lignin in nature. They can degrade a wide range of persistent aromatics and produce complex of extracellular nonspecific enzymes including Mn-peroxidases (EC 1.11.1.13), versatile peroxidases (EC 1.11.1.16), and laccases (EC 1.10.3.2). The ability of Pleurotus fungi to survive in soil, compete with the indigenous microflora, and degrade soil pollutants makes them promising candidates for mycoremediation [Pozdnyakova et al., 2017].

Bacteria belonging to the genus Azospirillum are free-living microbes that promote plant growth (PGPR). The most accepted theory regarding the mechanism of action of Azospirillum is its growth promotion, which includes nitrogen fixation and phytohormone (including IAA), polyamine, and trehalose production. There are reports on using Azospirillum strains in pollutant (phenol, benzoate, protocatechuate, catechol, phenanthrene, and crude oil) degradation process [Muratova et al., 2005; Cruz-Hernandez et al., 2022]. The production of laccase and ligninolytic peroxidases, like fungi, by Azospirillum are known too [Diamantidis et al., 2000; Kupryashina et al., 2015].

 

3. Line 87. Include reference to the gravimetric method

The growth of P. ostreatus mycelium was controlled by the weight method. The mycelium of the fungus at the end of the experiment was separated by filtration through pre-weighed paper filters. Filters with mycelium were dried to constant weight and weighed again. The weight of the mycelium was calculated from the difference between the weight of the filter and the weight of the filter with mycelium. We added the reference.

 

4. Line 98. Standardize the forms of entry of values and quantities, this is different from the previous paragraphs.

We standardized the forms of entry of values and quantities.

 

5. Line 100. The final total concentration of 5 mg per 100 ml of each PAH?

Not. Individual PAHs were added at 1 mg. Their total concentration was 5 mg/100ml.

 

6. Line 101. How many microorganisms were added?

In all experiments, the quantities of microorganisms were the same. Fungal mycelium (20 mg wet weight) was added as 5 ml of inoculum previously grown in basidiomycetes rich medium. The bacterium previously was grown on Petri dishes with R2A agar medium, washed off with physiological saline, and added to flasks up to 10^-3 CFU.

 

7. Line 119. What formula was used to arrive at U/ml? We have a serious problem with the incorrect use of formulas for this group of enzymes. We will only solve this problem by not omitting this information.

The standard formula for determination of enzymatic activity was used. This takes into account: the change in adsorption per minute, the volume of the reaction mixture, the molar extinction coefficient, the length of the optical way. Due to the impossibility to determine the protein concentration in cultivation medium accurately, we used arbitrary units [µmol/min/ml of enzyme preparation.

 

8. Has the adsorption of PAHs on the mycelium not been evaluated? We know that extraction with chloroform alone does not release material adsorbed by the hyphae. It is necessary to break the mycelium beforehand.

In the previous experiments, different methods of extraction of PAHs using different solvents were evaluated. Mycelium and culture medium were separated by filtration. PAHs were extracted from the culture medium. Mycelium (wet or dry in different treatments) was homogenized with quarz sand and then extracted with different solvents. In all treatments sorption of PAHs by fungal mycelium did not exceed 5-7%.

 

9. Line 126. Include the methodology in the previous part.

The study of antagonistic activity of Pleurotus ostreatus Florida and Azospirillum brasilense SR80 were the subject of separate work.  Since the data of these experiments is not presented in this article, we believe that it is not necessary to include the double culture technique to the section “Materials and Methods”.

 

10. Line 139. I think this conclusion is hasty at this point, without discussion.

The use of fungal PAH metabolites as a carbon source by bacteria is known (for example see Brodkorb, Legge Appl. Environ. Microbiol. 58 (1992) 3117-3121). According to the data of different authors, we proposed that Azospirillum brasilense SR80 can also utilize the products of degradation PAHs by Pleurotus ostreatus Florida. This hypothesis will be the subject of further research.

 

11. Line 143. As it seems that the mycelium did not break down, we cannot say that only the reduction in concentration is the result of degradation (figure 1), at this time of presentation of the results. After evaluating the metabolites, yes, we can say that there was degradation. However, not every % decrease in concentration can be the result of degradation. If the adsorption has not been evaluated, I suggest changing, at this point the use of the word degradation. Perhaps Disappearance like this in the picture.

We replaced “degradation” by “disappearance or utilization” at this stage, as you suggest.

 

12. Line Reference?

We added the reference.

 

13. Line 178. Make this experiment clearer in the methodology. Got confused

According to the obtained data, fluorene and its degradation product, 9-fluorenone, were chosen for further study of the metabolism by co-cultures. The chosen temperature of 30°C was optimal for the growth of both cultures.

 

14. Line 223. This goes into the methodology

We have removed duplicates.

 

15. Line 239. This entire part of the IAA was not presented in the method.

As I understand it, there was induction with exogenous IAA and the evaluation of IAA as a metabolic of microorganisms. That's it? How were these experiments?

We added explanation to Methods.

 

16. 4.1 Line 250 to 269: This is the introduction, not a discussion.

We added an explanation of the selection of Pleurotus and Azospirillum as research subject in the Introduction as you suggest. Here we discuss the possibility to use specific strains as a model to study PAH degradation under co-cultivation conditions.

 

17. Line 284. I believe it is not a valid reference. What is the difference between the cited work and the current work?

Tsivileva et al. showed the stimulating effects of PGPR bacteria on the growth rate, the formation of fruiting bodies and the productivity of edible mushrooms, including oyster mushrooms Pleurotus ostreatus. Our studies show the mutual influence of Pleurotus ostreatus Florida and Azospirillum brasilense SR80 during PAH utilization. The increase in the dry weight of mycelium was only one of the observed effects.

 

18. Line 304. It's the same information as the result. What's the discussion?

We changed this sentence and added a discussion.

 

19. 4 Line 311. Reference? These same retention times were found in other works in the literature. Even with other microorganisms? The discussion must go in that direction.

The retention times differ in different works, because often different columns and separation conditions are used. Therefore, we used commercial compounds to identify the parent PAHs and their metabolites. One from the key metabolites of PAH degradation by fungi is phthalic acid, which, through a number of stages, is included to the main metabolism. However, the retention times of the metabolites identified by us do not correspond to phthalic acid. They appeared on a later day of cultivation and disappeared with an increase in cultivation time. This may mean that they are products of a deeper degradation of PAHs. At this stage of the study, we were unable to identify these compounds. Their identification will be the subject of further research.

 

20. Line 317. “This may suggest the similar mechanisms in the initial attack of PAH molecules in the organisms studied.” Your work has no basis for this claim. What do the other works say? How is the metabolite pathway of each organism, and what are the intermediaries?

This is not a statement, but a working hypothesis that will be the subject of further research. The formation of the corresponding quinones at the first stage of PAH degradation by ligninolytic fungi is well known. The reactions of ligninolytic peroxidases and laccases lead to the formation of these products [Kadri et al., 2016; Ghosal et al., 2016]. In our experiments, the formation of quinone metabolites in fungal monoculture and co-culture with A. brasilense SR80 can also be the result of the catalytic action of laccase and hybrid peroxidase produces by this fungus. 9-Fluorenone revealed as product of bacterial degradation of fluorene can be an indirect confirmation of the participation of bacterial laccase in the degradation of this PAH. Currently, there are no studies on the metabolism of PAHs by Azospirillum, so we could not compare our results.

 

21. Line 321. Reference

We added the reference.

 

22. Line 335. In this sentence and others, the scientific names are not in italics. Review.

We italicized all names of organisms through the text.

 

23. Line 343. What is the IAA? Include in the introduction and emphasize the importance of the discussion

Some authors believe that indole-3-acetic acid (IAA), which is a phytohormone of the auxin class, can also be a universal physiological code not only for the plants but also for bacteria and fungi. Increasing evidence shows IAA as a diffusible signal that is used for interspecies communications. This phenomenon suggests the existence of a framework, widely evolved in both eukaryotes and prokaryotes, allowing the production, transfer, and perception of IAA signals between distantly related organisms across the branches of phylogenetically diverse groups of the tree of life [Fu et al., 2015]. The production of IAA by bacteria is well studied. Although, IAA-producing fungi are known, its biosynthetic pathways and role in fungal ecology have not been widely studied. We added this information to Introduction.

 

24. Is the addition of IAA interesting for a bioremediation process?

Yes, we believe that the study of the effects of IAA may be important for bioremediation processes. The positive effect of IAA on mycelium growth can contribute to better survival of the fungus under conditions of mycoremediation. Of course, it is not advisable to introduce exogenous IAA into the soil. However, the combined use of a fungus and IAA-producing bacterium, which also has degradative properties, can accelerate the remediation of PAH-contaminated soils. This will also be the subject of further research.

 

25. 10 Many studies have shown that in some cases the metabolites generated in the degradation process are more toxic. There are simple experiments that must be carried out by the group to really confirm the potential of the proposed study.

It is true that some metabolites may be more toxic than the parent compounds. We have done a similar study [Pozdnyakova, N.; Dubrovskaya, E.; Schlosser, D.; Kuznetsova, S.; Sigida, E.; Grinev, V.; Golubev, S.; Kryuchkova, E.; Varese, G.C.; Turkovskaya, O. Widespread ability of ligninolytic fungi to degrade hazardous organic pollutants as the basis for the self‐purification ability of natural ecosystems and for mycoremediation technologies. Appl. Sci. 2022, 12, 2164.

https://doi.org/10.3390/app12042164; Dubrovskaya E.V., Pozdnyakova N.N., Muratova A.Yu., and Turkovskaya O.V. Changes in phytotoxicity of polycyclic aromatic hydrocarbons in the course of microbial degradation // Russian Journal of Plant Physiology. – 2016. – Vol. 63, No. 1. – P. 172–179. https://doi.org/10.1134/S1021443716010052]. Phytotests showed that the effects of PAHs and their derivatives agree with aqueous solubility of the tested compounds. PAHs, as poorly soluble compounds, were less phytotoxic than their oxidized derivatives. Oxidized PAH metabolites presented by quinones, ketones, and acids are more soluble in water than PAH; therefore, their phytotoxicity can be greater. However, there is a possibility of PAH mineralization to CO₂ and H₂O by the studied fungus and its co-culture, as was shown for other fungi. Therefore, at the next stage, we plan to study in detail the characteristics of PAH metabolism by the co-culture.

 

26. 11 Correct the scientific names of the figures

The scientific names of the figures were corrected.

 

27. 12 The discussion should be further worked on. I highlight the lack of discussion of metabolites and their importance in the decontamination process.

We added discussion of metabolites formed through text.

Reviewer 2 Report

This study investigated the synergistic/ antagonistic effects of the co-culture of fungi and bacteria on polycyclic aromatic hydrocarbon degradation. The manuscript was well-written and organized. The obtained data was sufficiently interpreted and discussed. Some minor points should be revised before publication.

- Line 84: should be "Fugus was grown on the modified medium.."

- Fig 1, 2, 3: Y axis should be "Removal (%)" or "Degradation efficiency (%)" instead of " Disappearance(%)"

- Fig 5, X-axis: microorganism's name should be italicized.

Author Response

1. Line 84: should be "Fugus was grown on the modified medium.."

We corrected this sentence as you suggest.

 

2. Fig 1, 2, 3: Y axis should be "Removal (%)" or "Degradation efficiency (%)" instead of " Disappearance(%)"

According to recommendation of rReviewer 1, we used the term “disappearance” on the figures and in the text.

 

3. Fig 5, X-axis: microorganism's name should be italicized.

We corrected it as you suggest.

Reviewer 3 Report

The manuscript ‘Degradation of polycyclic aromatic hydrocarbons by dual cultures of Pleurotus ostreatus Florida and Azospirillum brasilense’ involves an interesting approach to degrade polycyclic aromatic hydrocarbons by using a coculture system of Pleurotus ostreatus Florida and Azospirillum brasilense. The manuscript, however, requires a thorough grammar check, editing and proofreading. It is recommended for publication in the applied microbiology journal with minor revisions.

If laccase and peroxidases are absent in the bacterial species, what causes the degradation?

Line 159 Despite the active growth of A. brasilense the bacterial degradation of PAHs was lower at 37ºС. Why?

Can you substantiate why pyrene degradation is lower at 30 ºС compared to 24 ºС?

The authors might discuss the research gap and future research directions at the end of the manuscript.

Minor comments:

The phrase ‘dual culture’ doesn’t sound appropriate. Change it to coculture or something more suitable.

Rewrite sentences 52, 57, 156, 177, 195, 245, 348, 356 for better understanding.

Move lines 109 to 111 to the results section

Line 122 replace ‘processing’ with ‘analysis’

‘temperature of …’ Statement incomplete in line 157

A. brasilense is not italicized in some places.

Line 210 ‘of’ missing

Microbes are not italicized between lines 333 to 343

Consider replacing ‘assumption’ with ‘hypothesis’ as it is repetitive.

Line 359: Replace ‘along’ with ‘through’

 

 

 

Author Response

1. If laccase and peroxidases are absent in the bacterial species, what causes the degradation?

More often in bacterial degradation pathways, the PAHs is activated by incorporating molecular oxygen directly with the help of mono- or dioxygenases [Srivatstava, Kumar 2019]. However, the bacteria producing laccase and ligninolytic peroxidases, like fungi, are known too [Diamantidis et al., 2000]. We added this information to Introduction.

 

2. Line 159 Despite the active growth of brasilense the bacterial degradation of PAHs was lower at 37ºС. Why?

We studied the degradation of PAHs under co-metabolism. We can assume that the temperature of 37°С is more suitable for the utilization of the growth substrate (glucose, fructose) than PAHs. Therefore, we observe more active growth, but less active utilization of PAHs.

 

3. Can you substantiate why pyrene degradation is lower at 30 ºС compared to 24 ºС?

The main contribution to the degradation of PAHs seems to be made by extracellular enzymes of the fungus. They are less stable at a temperature 30 ºС than at the temperature of 24 ºС.

 

4. The authors might discuss the research gap and future research directions at the end of the manuscript.

We added the research gap and future research through text.

 

5. Minor comments: The phrase ‘dual culture’ doesn’t sound appropriate. Change it to coculture or something more suitable.

Both terms “dual culture” and “co-culture” are used in the literature (Hatvani et al., J. Appl. Microbiol. 92 (2002) 415-423; Zhang et al., Appl. Microbiol. Biotechnol. 73 (2006) 89-94; Chi et al., Int. Biodeterior. Biodegrad. 59 (2007) 32-39; Flores et al., J. Appl. Microbiol. 106 (2009) 249-257; Kumari, Naraian J. Environ. Management 180 (2016) 172-179). However, as you suggest we replaced “dual culture” by “co-culture” through text.

 

6. Rewrite sentences 52, 57, 156, 177, 195, 245, 348, 356 for better understanding.

The English was corrected through text.

 

7. Move lines 109 to 111 to the results section

We've added clarifications to the Methods part.

 

8. Line 122 replace ‘processing’ with ‘analysis’ ‘temperature of …’ Statement incomplete in line 157 brasilense is not italicized in some places.

We corrected it.

 

9. Line 210 ‘of’ missing

We corrected it. It was a technical error.

 

10. Microbes are not italicized between lines 333 to 343

We italicized the names of the microorganisms through text.

 

11. Consider replacing ‘assumption’ with ‘hypothesis’ as it is repetitive.

We changed this sentence by “To test whether IAA affected growth…”.

 

12. Line 359: Replace ‘along’ with ‘through’

We changed this sentence by “…..by way of the minor pathway….”.