Biosurfactant Production from the Biodegradation of n-Paraffins, Isoprenoids and Aromatic Hydrocarbons from Crude Petroleum by Yarrowia lipolytica IMUFRJ 50682

Round 1

Reviewer 1 Report

Manuscript Title: Biosurfactant production from the biodegradation of n-paraffins, isoprenoids and aromatic hydrocarbons from crude petroleum by Yarrowia lipolytica IMUFRJ 50682.

I found the manuscript interesting. Not many organisms are capable of degrading both aliphatic and aromatic hydrocarbons.  But several issues should be addressed, before the manuscript can be published.

1. This specific strain was identified a long time ago. Are any new data (gene or genome sequences) publicly available?

2. I do not think that the spectrophotometric method is the best choice for TPH determination. For quantitative purposes GC-FID or FTIR should have been used.

3. Does the percentage of oil removal refers to the original crude oil or oil extracted from the abiotic control? It seems that abiotic losses are also significant.

4. The biomass in samples containing crude oil as a sole carbon source seems was quite stable (Fig.1), which is absolutely OK, since only a limited number of cells could attach to oil. Therefore, it is unlikely that the total cell number reached such an unbelievably high value (1.3x10¹⁴ CFU/mL, Table 1 ). My opinion is supported by the photographs shown (Figs.2 and 10). In fact, even the initial value (3.7x10¹² CFU/mL) seems to be far too high (at least three orders of magnitude).  Perhaps the presented values refer to one L and not mL? This definitely needs to be corrected.

5. Section Materials and Methods, line 117. What was the reason for protease determination? There is no single result concerning this particular enzyme.

6. The results concerning glucose consumption (Fig.1) are trivial. I would remove this graph.

7. Can the Authors identify at least a few peaks presented in Fig 3?

8. No statistical analysis is presented.  For example it is not clear whether the biomass increase presented (Fig.1) was the result of  oil degradation.

Author Response

I found the manuscript interesting. Not many organisms are capable of degrading both aliphatic and aromatic hydrocarbons.  But several issues should be addressed, before the manuscript can be published.

Response: We thank the reviewer for the positive evaluation and for the suggestions that increased the quality of the manuscript.

Point 1: 1. This specific strain was identified a long time ago. Are any new data (gene or genome sequences) publicly available?

 Response 1: No, this specific strain has never been sequenced. We have only performed a Automatic sequencing by capillary electrophoresis in the ABI 3730xl DNA Analyzer equipment (Applied Biosystems) and alignment of the nucleotide sequences produced with the reference sequences deposited in GenBank. The sequenced microorganism showed 99.46% identity with the fungus species Yarrowia lipolytica (accession number KY105975.1). This data has not been published.

 

Point 2: 2. I do not think that the spectrophotometric method is the best choice for TPH determination. For quantitative purposes GC-FID or FTIR should have been used.

Response 2: Yes, we agree with the reveiwer, but the spectrophotometric method was only used as a first and fast evaluation of hydrocarbon consumption (comparing to the abiotic control). The main goal in this study was not to analyze the exact amount that was biodegraded but what type of compounds were consumed, which was then analyzed by GC-MS.

 

Point 3: 3. Does the percentage of oil removal refers to the original crude oil or oil extracted from the abiotic control? It seems that abiotic losses are also significant.

 Response 3: The percentage of oil removal refers to the oil extracted from the abiotic control. In order to make it clear, the sentence “The percentage of oil removal was calculated with the abiotic experiment as a control.”was added to section 2.3.

Point 4: The biomass in samples containing crude oil as a sole carbon source seems was quite stable (Fig.1), which is absolutely OK, since only a limited number of cells could attach to oil. Therefore, it is unlikely that the total cell number reached such an unbelievably high value (1.3x10¹⁴ CFU/mL, Table 1 ). My opinion is supported by the photographs shown (Figs.2 and 10). In fact, even the initial value (3.7x10¹² CFU/mL) seems to be far too high (at least three orders of magnitude).  Perhaps the presented values refer to one L and not mL? This definitely needs to be corrected.

 

Response 4: CFU determination was repeated for the initial sample in duplicate and we have confirmed the value in Table 1. The new results were 1.01 x 10¹² CFU/mL (101 cells in a plate from a dilution of 10^-9, with 0,1 mL in the plate) and 2.32 x 10¹¹ CFU/mL (232 cells in a plate from a dilution of 10^-8, with 0,1 mL in the plate). So, we have calculated the average value (using the three results), and this is the new value in Table 1 (1.65 x 10¹²). The initial cell concentration of the experiments is 1 g of cells per liter, which is high indeed. 

Point 5: Section Materials and Methods, line 117. What was the reason for protease determination? There is no single result concerning this particular enzyme.

 Response 5: Yes, the reviewer is right. This was a mistake and we have removed protease from line 117.

Point 6: The results concerning glucose consumption (Fig.1) are trivial. I would remove this graph.

 

Response 6: We agree with the reviewer and the glucose consumption graphic was removed.

Point 7: Can the Authors identify at least a few peaks presented in Fig 3?

 Response 7: Chromatograms presented in Fig. 3 have hundreds of peaks and most of them are overlapped since the total time analysis was long and the chromatograms were reduced to fit manuscript. Identifying the peaks was not the purpose of this figure although we can easily identify Pristane and Phytane which are the longer peaks around 21 and 22.5 minutes, respectively.

Point 8: 8. No statistical analysis is presented.  For example it is not clear whether the biomass increase presented (Fig.1) was the result of  oil degradation.

Response 8:  Standard deviation of the data was added to the results of Fig. 1.

Author Response File: Author Response.docx

Reviewer 2 Report

The science presented is original with plenty of detail. In general, the authors have done a good job explaining the background information necessary to appreciate the rationale and results of the experiments. The MS is well written. There are no objective errors in the methods and results. However, some minor amendments are needed. Conclusions are a little poorly written.

Author Response

General aspects: The science presented is original with plenty of detail. In general, the authors have done a good job explaining the background information necessary to appreciate the rationale and results of the experiments. The MS is well written. There are no objective errors in the methods and results. However, some minor amendments are needed.

 Response: We thank the reviewer for the positive evaluation and for the suggestions that increased the quality of the manuscript.

 

Point 1: Conclusions are a little poorly written.

 Response 1: We agree with the reviewer and the conclusion section has been revised.

 

Author Response File: Author Response.docx

Round 2

Reviewer 1 Report

The Authors have addressed most of the issues raised. But my two concerns are still present.

1. The presented  CFU values are definitely far too high. It is impossible to reach such high densities, especially when the only available carbon source is hydrophobic. Please refer to your own data. There was virtually no change in biomass when the cells were cultivated with crude oil only (Fig. 1); therefore no significant change in cell number should have been obtained. But this is even less important. How come that high number of cells (1.3x10¹⁴ CFU) could have “fitted” within 1 ml of medium? What is an average size of yeast cell? Please also look at Fig 10. It seems that culture medium was not fully packed with Yarrowia cells. Something must be wrong with calculations, even corrected initial value seems to be incorrect (the mean of 1.01 x 10¹² CFU/mL and 2.32 x 10¹¹ CFU/mL is not 1.65 x 10¹² CFU/mL). The only logical explanation which comes to my mind is the all values are expressed as CFU/L instead per mL.

2. No statistical analysis is presented. It is still not clear whether the biomass increase presented (Fig.1) was the result of  oil degradation.

Author Response

The Authors have addressed most of the issues raised. But my two concerns are still present.

Response: We thank the reviewer for the positive evaluation and for the suggestions that increased the quality of the manuscript.

Point 1_1: 1. The presented CFU values are definitely far too high. It is impossible to reach such high densities, especially when the only available carbon source is hydrophobic. Please refer to your own data. There was virtually no change in biomass when the cells were cultivated with crude oil only (Fig. 1); therefore, no significant change in cell number should have been obtained. But this is even less important.

Response 1_1: Figure 1 refers to the turbidimetric method. Samples from the aqueous phase are taken, and absorbance is measured. Since many cells have adhered to the oil, these cells are not quantified by this method.

Point 1_2: How come that high number of cells (1.3x10¹⁴ CFU) could have “fitted” within 1 ml of medium? What is an average size of yeast cell?

Response 1_2: The reviewer is completely right. The average size of this yeast is 5 µm. Considering this value, the diameter of the cell (as an sphere), a maximum of 10¹⁰ cells would fit into 1 mL.

Point 1_3: Please also look at Fig 10. It seems that culture medium was not fully packed with Yarrowia cells.

Response 1_3: These are diluted samples.

Point 1_4: Something must be wrong with calculations, even corrected initial value seems to be incorrect (the mean of 1.01 x 10¹² CFU/mL and 2.32 x 10¹¹ CFU/mL is not 1.65 x 10¹² CFU/mL).

Response 1_4: “So, we have calculated the average value (using the three results),” This average would be (1.01 x 10¹² CFU/mL + 2.32 x 10¹¹ CFU/mL + 3.7 x 10¹² CFU/mL)/3. But the reviewer is right there was something wrong with the values, and new experiments were performed to verify this data.

Point 1_5: The only logical explanation which comes to my mind is the all values are expressed as CFU/L instead per mL.

Response 1_5: The reviewer is completely right. The experiment was repeated in triplicate for all conditions, and the results confirmed that the CFU/mL was wrong. The new values and the statistical analysis of them are reported in Table 1.

Point 2:2. No statistical analysis is presented. It is still not clear whether the biomass increase presented (Fig.1) was the result of  oil degradation.

Response 2: An statistical analysis has been performed, and the methodology of this analysis was included in the methodology section (section 2.3).

 

Author Response File: Author Response.docx

Round 3

Reviewer 1 Report

Dear Authors,

Thank You very much for addressing all issues. The only thing which requires correction is p value which should be < 0.05 or < 0.01 (line 172). The manuscript can be published. 

Author Response

Thank You very much for addressing all issues. The only thing which requires correction is p value which should be < 0.05 or < 0.01 (line 172). The manuscript can be published. 

Response: The p value has been changed for 0.05 in line 172.

Author Response File: Author Response.docx