The Potential Applications of Bacillus sp. and Pseudomonas sp. Strains with Antimicrobial Activity against Phytopathogens, in Waste Oils and the Bioremediation of Hydrocarbons
, Nicoleta Ene 2,4, Nereida Malo (Dalanaj) 5, Ovidiu Popa 1 and Narcisa Babeanu 1Round 1
Reviewer 1 Report
The manuscript has been improved according to the indication given. Few spelling mistakes are still present and I cannot judge the final quality of the figures, but some of them are very small in my file, expecially the graph axes numbering. Nonetheless I guess this can be fixed in the editing process of the draft.
Author Response
Thank you very much for your rightly observations!
We made the corrections of the figures in the revised manuscript. We kindly ask you to see our changes.
Reviewer 2 Report
The manuscript needs to undergo major improvement in its technical content. No remediation of organics was in fact attempted. Emulsification capacity is not equivalent to remediation. The English of the manuscript needs improvement.
Experimental work pertaining to the current study should be at least briefly explained. Reference can be made to the literature for more details. How was the emulsification index calculated?
Author Response
Comments and Suggestions for Authors
The manuscript needs to undergo major improvement in its technical content. No remediation of organics was in fact attempted. Emulsification capacity is not equivalent to remediation. The English of the manuscript needs improvement.Thank you very much for your observations!
We hope we improved the revised version.
Is true that emulsification capacity is not equivalent to remediation but many researchers have established direct connections between the emulsification capacity of a biosurfactant and bioremediation. The biosurfactants that registered good emulsification indices, proved to be most capable to make the insoluble substrate accessible for microbial biodegradation (Desai and Banat, 1997; Chandankere et al., 2014; Silva et al., 2018).
The revised manuscript shows a substantial extensive English corrected version. Experimental work pertaining to the current study should be at least briefly explained.
You are right! We filled out the necessary explanations on conducting experiments. We kindly ask you to find them in the chapter “Materials and Methods”.
Reference can be made to the literature for more details.
We also provided the necessary references.
How was the emulsification index calculated?
The emulsification index was calculated with formula:
E24 = (Height of emulsion layer/Height of total liquid column) x 100
The formula is given in the chapter “Materials and Methods”, sub-chapter “Emulsification index test”.
Reviewer 3 Report
In this paper the Potential Applications of Bacillus sp. and Pseudomonas sp. strains are described.
Introduction:
line 43/54: I recommend writing in italics Bacillus, Pseudomonas, Rhodococcus....
It might be useful to specify which types of biosurfactants are present in the literature (what kind of molecules).
line 59: you can specify which types of microorganisms and, if known in literature, the mechanism of action.
Materials and Methods
line 90: it would be interesting to provide some indication of the protocol used
line 126: In this part of your study, have you used the same soil for bacteria and fungi?
If instead you had already isolated the two stocks of interest, you need to specify it.
line 143: If you do not explain how you have added the phytopathogen it is difficult for the reader to understand and repeat the experiment. You have to describe the protocol.
line 146: For the reader it would be clearer to explain in detail what you mean by antagonist
line 149: specify the concentration of 1ml of the phytopathogen suspension.
line 156: specify temperature and rpm.
line 159: Are you sure that this type of sterilization does not involve a variation in the molecules of interest?
line 166-172: I think this part should be included in the part of results and discussion
Results and discussion
Graphs 1-6: to better understand the pH variations a secondary axis could be inserted in the graphs
Table 1-2: if you have conducted experiments in triple you should enter the error values
You have described in detail the part concerning the emulsification index, you could do the same for the part concerning the enzymes.
For completeness of the work it would be useful to quantify the enzymatic activity of amylase, catalase and lipase of these microorganisms.
Author Response
Thank you very much for your observations! Please find our changes below:
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No. |
Reviewer Comments and Suggestions for Authors
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Author’s Response |
Revised Text |
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1 |
Introduction line 43/54: I recommend writing in italics Bacillus, Pseudomonas, Rhodococcus....
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As you suggested, we made the modification. |
Line 46 : Many of the bacteria belonging to Bacillus and Pseudomonas genera...
Line 56: Pseudomonas, Bacillus, Rhodococcus and Candida genera.. |
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2 |
Introduction It might be useful to specify which types of biosurfactants are present in the literature (what kind of molecules). |
In revised version we specified the types of biosurfactants. |
Line 58 : Having different molecular structures and surface activities the biosurfactants are divided in different classes: glycolipids... |
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3 |
Introduction line 59: you can specify which types of microorganisms and, if known in literature, the mechanism of action.
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Please find all the information in the revised version. |
Line 67 : According to some researchers, the production of peptides with antimicrobial activity... Line 69 : These peptides, commonly known as lipopeptides, are capable to damage the plasma membrane of the target microorganisms...
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4 |
Materials and Methods line 90: it would be interesting to provide some indication of the protocol used |
We hope we have provided all the information now. |
Line 265 : The plant materials were collected from different areas of Romania (Valcea, Bucharest, Ilfov) in sterile recipients... |
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5 |
Materials and Methods line 126: In this part of your study, have you used the same soil for bacteria and fungi? If instead you had already isolated the two stocks of interest, you need to specify it. |
As you suggested, we made the specifications. |
Line 303 : The phytopathogen strains used in experiments, X. campestris ICCF 274 and E. carotovora ICCF 138, are from... |
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6 |
Materials and Methods line 143: If you do not explain how you have added the phytopathogen it is difficult for the reader to understand and repeat the experiment. You have to describe the protocol. |
We hope we have provided the necessary information. |
Line 324 : The first experiment (Experiment I) followed an approach described by Soare et al.: one ml of inoculum from the broth culture of phytopathogens (containing 3x108 UFC/mL, McFarland Standard No. 1) was added by pipette to the center of the Petri dish, over the agar medium (cooled, but still molten) and rotated gently, till they mixed... |
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7 |
Materials and Methods line 146: For the reader it would be clearer to explain in detail what you mean by antagonist |
We hope we have provided the necessary information. |
Line 42 : These antagonists, also known as biocontrol agents are commonly occurring nonpathogenic microbes such as fungi, bacteria or yeast. Antagonists act directly on phytopathogens through different mechanisms... |
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8 |
Materials and Methods line 149: specify the concentration of 1ml of the phytopathogen suspension. |
Please find all the information in the revised version. |
Line 332 : In the second experiment (Experiment II), the phytopathogens (1 ml of inoculum from the broth culture of phytopathogens, containing 3x108 UFC/mL) were first added to the culture medium according to the same method. |
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9 |
Materials and Methods line 156: specify temperature and rpm. |
Please find the information in the revised version. |
Line 346 : For the antimicrobial activity assay of the biosurfactants, the strains of B. mycoides (Bm) and P. putida (B1) were grown submerged, for 72 hours, at 28-30°C and 220 rpm, on five media... |
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10 |
Materials and Methods line 159: Are you sure that this type of sterilization does not involve a variation in the molecules of interest? |
We hope we have provided the necessary information. |
Line 349 : The supernatants used in experiments were sterilized for 30 minutes at 115°C and after sterilization no significant changes were registered in the emulsification activity. Many authors mention that some biosurfactants are heat resistant [41] (Nitschke and Pastore, 2006), while some are not... |
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11 |
Materials and Methods line 166-172: I think this part should be included in the part of results and discussion |
As you suggested, we made the modification. |
Line 180 : Extracellular enzymes and metabolites production are often associated with antagonism properties [28] (Nielsen et al., 2002). Most of the species of Bacillus and Pseudomonas genera, have the ability to produce extracellular enzymes... |
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12 |
Results and discussion Graphs 1-6: to better understand the pH variations a secondary axis could be inserted in the graphs |
As you suggested, we made the modification. |
Line 121-152 : Figure 1....Figure 5 |
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13 |
Results and discussion Table 1-2: if you have conducted experiments in triple you should enter the error values |
As you suggested, we made the modification. |
Line 166-171 : Table 1, Table 2 |
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14 |
Results and discussion You have described in detail the part concerning the emulsification index, you could do the same for the part concerning the enzymes. For completeness of the work it would be useful to quantify the enzymatic activity of amylase, catalase and lipase of these microorganisms. |
Please find the information in the revised version. |
Line 192 : The enzymatic activities measured for amylase (Enzymatic Assay of α-Amylase EC 3.2.1.1, with DNS), lipase (Enzymatic Assay of LIPASE EC 3.1.1.3, titrimetric method) and catalase (Enzymatic Assay of Catalase EC 1.11.1.6), following the Sigma-Aldrich protocols revealed that: - after 72 hours of development in SNA medium B. mycoides (Bm) registered 0.75 U/ml and the amylase activity of P. putida (B1) strain was insignificant. - after 48 hours of development in LA medium B. mycoides (Bm) registered 0.39 U/ml and the lipase activity of P. putida (B1) strain registered 0.36 U/ml. - after 72 hours of development on M44 solid medium P. putida (B1) registered 2,3 U/ml and the catalase activity of B. mycoides (Bm) strain was insignificant. |
Round 2
Reviewer 2 Report
The current format reads much better and properly referenced.
Reviewer 3 Report
You have applied all the corrections to the observations I had made to you
This manuscript is a resubmission of an earlier submission. The following is a list of the peer review reports and author responses from that submission.
Round 1
Reviewer 1 Report
The manuscript contains potentially some interesting data but there are major flaws in the presentation of data and in the overall organisation of the manuscript.
The data presentation problems are mainly linked to the lack of error bars in all the graphs. The authors should provide the errors on their measurements. Also many results are presented and discussed in a qualitative style and not in a quantitative format.
I cannot find any indication on how the strains were assigned to the Genera and the strain indication are quite generic. The autghors also missed to mention in their introduction that there are other Genera wit the same features (as an example the Genus Acinetobacter is also producing surfactants).
The overall organisation and title of the manuscript is misleading: the data presented are on two novel bacterial isolates from plants, not selected on any pollutants, and there is a very far-fetched link to the mentioned wasted oils and hydrocarbons bioremediation: the isolates were selected on the basis of antimicrobial activity but the emulsification index tests are very clearly and logically linked (at least in the discussion) to the antimicrobial activity.
Although I can understand in principle the connections proposed by the authors and the results on the emulsification index on heptane and octane if the Bacillus is quite promising, the manuscript is very obscure and difficult to follow in this respect. The phytopatogens control aspect is mentioned in the text but not coherently with the title: I suggest to deeply revise the title and the introduction.
The most interesting data are in the emulsifying properties, but there is no comparison to any strain in literature and non control provided in the experiments. the authors should integrate on this point.
As a minor point I do not understand the title of the Material and Methods section: 2.1. Formatting of Mathematical Components
There is also a mistake in a legend (Figure 15) referring to octane, while the data are clearly indicated as results on Heptane.
Reviewer 2 Report
In the paper entitled “Potential applications of some newly isolated microorganisms in wasted oils and hydrocarbons bioremediation”, the authors have studied the production of compounds active in waste oils and hydrocarbons bioremediation starting from new isolated microorganisms. Despite the topic is very interesting the manuscript has many problems.
In some parts the paper seem to be very confused: for example, in the text the figures and tables are not indicated, making very difficult to follow the text without these indications. The letters that indicate the panels of figures 6 and 7 are partially deleted; the panels of figure 7 do not start as A,B but are reported as C,D. In every part of the paper the name of bacteria is not indicated in italic font; the English style needs to be completely revised.
Below are reported some corrections:
- page 1, line 20: it is reported “In this study, obtained results…”, probably it is better change in “In this study are reported results..”.
- page 1, line 36: “hy-drocarbons”, please correct it in “hydrocarbon”.
- page 1, line 38: at the end of sentence is missing the point.
- page 2, line 99: “28-30oC”, please correct it in “28-30°C”.
- page 2,line 100: “9*108”, do you mean “9x108”?
- page 2, lines 106-107: “.. for their antimicrobial activity. The antimicrobial activity assay was …”, please change as “.. for their antimicrobial activity, the assay was….”.
- page 2, line 118: “9*108”, do you mean “9x108”?
- page 2, line 120: “2*d”, do you mean “2xd”?
- page 3, line 148: is there a control for this experiment? Is it only a qualitative method?
Figures 6, 7, 8 and 9 give an idea of experiments done in a not rigorous way, in may opinion adequate and rigorous control are lacking (supported by numbers, error bars, negative and positive controls, ie bacteria that have or not antimicrobial and biosurfactant production).
For these reasons the manuscript must be rejected.
Best Regards
