New Biotechnological Production of EPA by Pythium irregulare Using Alternative Sustainable Media Obtained from Food Industry By-Products and Waste
, Antonio Luca Langellotti 1, Beatriz MartÃn-GarcÃa 2,3, Vito Verardo 2,3, Raffaele Romano 4, Raffaele Sacchi 4 and Paolo Masi 1,4
Abimbola E. Oluwalana
Round 1
Reviewer 1 Report
Dear editor and authors, after a careful evaluation of the manuscript entitled “New biotechnological production of EPA by Pythium irregulare 3 using alternative sustainable mediums obtained from food in4 dustry by-products and waste”, I would like to recommend the present study to be accepted in this current form. The presented manuscript is well written and provide a good overview on some key production of EPA by Pythium irregulare aspects using a green alternative. The topic of this review is globally relevant and should be of interest for a broad readership
Author Response
Dear referee,
The authors wants to thank you for your time and considerations.
We are very glad that you liked the paper and that you found it interesting.
Best regards.
Reviewer 2 Report
The following report is based on my review of the manuscript entitled “New biotechnological production of EPA by Pythium irregular using alternative sustainable mediums obtained from food industry by-products and waste”, with manuscript ID sustainability-2120060. The manuscript fits within the scope of sustainability Journal and is also interesting and research is impressive. However, the following shortcomings have been pointed out and need to be addressed properly for further improvement of the manuscript. They are:
Ø Abstract did not captured the required date, kindly update.
Ø Irregularity in writing decree Celsius (x oC or oC) the best way is X oC. Please consider a space between and no underline for the (o).
Ø In your introduction need to be updated, kindly use this articles as a guide
doi.org/10.3390/pr10112262
10.1016/j.envres.2022.114091
doi.org/10.3390/separations9090258
Ø Conclusion is not enough.
Ø Figures are not clear, kindly provided high quality Figures.
Ø Figure 5 are not mentioned in the main text
Ø In line 390, number of Figure are not mentioned.
Ø Finally, the manuscript should be modified according to above said comments and be thoroughly reviewed again before accepting it for publication.
Author Response
Dear referee,
The authors would like to thank you for your time and comments to our manuscript.
I'll answer point by point (answer in bold):
Ø Abstract did not captured the required date, kindly update.
R: we corrected the abstract
Ø Irregularity in writing decree Celsius (x oC or oC) the best way is X oC. Please consider a space between and no underline for the (o).
R: we are sorry for the errors, we fixed as you suggested.
Ø In your introduction need to be updated, kindly use this articles as a guide
doi.org/10.3390/pr10112262
10.1016/j.envres.2022.114091
doi.org/10.3390/separations9090258
R: we added the suggested references to the manuscript
Ø Conclusion is not enough.
R: we enriched the conclusion with a new statement.
Ø Figures are not clear, kindly provided high quality Figures.
R: we provided 600 DPI figures, a transposition error probably occurred during the submission, we hope they are fine now.
Ø Figure 5 are not mentioned in the main text
R: we mentioned and discussed the Figure 5 in the paragraph "3.4 screening of food waste medium"
Ø In line 390, number of Figure are not mentioned.
R: we fixed this in the manuscript
Ø Finally, the manuscript should be modified according to above said comments and be thoroughly reviewed again before accepting it for publication.
R: We reviewed the manuscript according to your suggestions, and following also the suggestions of the other referees, we hope it is now suistable for publication.
Reviewer 3 Report
New biotechnological production of EPA by Pythium irregulare using alternative sustainable mediums obtained from food industry by-products and waste
Giovanni Luca Russoa, Antonio Luca Langellotti, Beatriz Martín-García , Vito Verardo, Raffaele Romano, Raffaele Sacchi, Paolo Masi
This study reports on the results obtained in the screening of by-products and wastes as nutrients for the cultivation of Pythium irregulare. The concentration of the by-product/waste selected as sources of C and N for maximizing the biomass concentration was determined using the Surface Response Methodology.
In order to be considered for publication an explanation is needed for the use of the biomass concentration as the response when the objective was the production of the long-chain omega 3 polyunsaturated, eicosapentaenoic acid (EPA).
It would be also of interest to compare the EPA yield with values reported for other microorganisms.
Other comments are
Line 27: change content by concentration.
Line 52: which is the average lipid content of the biomass?
Line 79: which is the composition of the standard medium?
Line 81-82: not sure the meaning of this phrase; was the EPA production studied? how?
Line 91: which is the composition of the V-8 Juice medium?
Line 99: what is the modified YEP medium?
Line 115: what was the concentration of yeast extract in these culture experiments?
Line 120: was glucose used as carbon source in these experiments? At what concentration?
Line 141: what was the chemical composition of the food by-products and waste used? Although these are used as sources of C or N, they also contain sugars, carbohydrates and nitrogen (as proteins and peptides).
Line 232: in the experiment with lactose, was residual galactose detected?
Line 232: what was the effect of the C source on the EPA synthesis and lipid content of the biomass?
Line 249: what was the effect of glucose concentration on the EPA synthesis and lipid content of the biomass?
Line 253: the quality of the graphs should be improved.
Line 254: caption to the figure is not self explanatory; which was the carbon source used? What was the concentration? What was the concentration of the different N sources? In graph B, what was the N source?
Line 262-264: this explanation is not clear.
Line 267: amino acids synthesis is an energy demanding process and microorganism save this energy using preformed amino acids. Besides amino acids are sources of both carbon and nitrogen.
Line 271-274: this explanation is not clear.
Line 280: which was the N source?
Line 287: N sources cannot be compared in terms of N content.
Line 288: what was the effect of the N sources and concentration on the EPA synthesis and lipid content of the biomass?
Line 306: a low temperature affects the kinetic of the process, and productivity instead of yield is decreased.
Line 313: for the conditions used in this work, what was the effect of the incubation temperature on the EPA synthesis and lipid content of the biomass?
Line 325: for the conditions used in this work, what was the effect of salinity on the EPA synthesis and lipid content of the biomass?
Line 350-351: which was the food by-product/waste that contained NaCl?
Line 357: which was the sugar composition of the juice?
Line 381: please check comas and symbols in the equation
Line 395: please explain why the inhibition to sporulation would reduce growth performance.
Line 406: if the objective of the study was the production of EPA then why EPA concentration was not used as the response for the optimization?
Line 442-444: please explain; it is not clear the relation of this text with the experimental results.
Line 452: which is the contribution of EFJ (used as source of sugars) and SBY (used as source of N) in terms of nitrogen and carbon, respectively?
Author Response
Dear referee,
The authors would like to thank you for your time and for all the comments/suggestions to our manuscript.
I'll answer point by point (answer in bold):
"In order to be considered for publication an explanation is needed for the use of the biomass concentration as the response when the objective was the production of the long-chain omega 3 polyunsaturated, eicosapentaenoic acid (EPA)."
R: The main aim of our study was to evaluate the growth of P. irregulare using alternative food waste-based mediums. To achieve that, we first had to evaluate the ideal growth conditions (since they were missing in the scientific literature). After that, we studied the lipid productivity and the EPA yield by comparing the food waste medium with the standard control, in order to evaluate if the new medium could have lipid yields worse than the standard medium.
"It would be also of interest to compare the EPA yield with values reported for other microorganisms."
R: we added some comparisons with other microorganisms in the section 3.6
Other comments are
Line 27: change content by concentration.
R: we fixed this in the manuscript
Line 52: which is the average lipid content of the biomass?
R: The average lipid content is 15-20% of DW, we specified this in the manuscript
Line 79: which is the composition of the standard medium?
R: The basal medium composition used was the modified YEP medium with the following composition: 20 g/L of glucose, 2 g/L of yeast extract, 2 g/L of peptone, 200 mg/L of K2HPO4, 5 mg/L of Fe-EDTA and 1 mL/L of A5 trace element solution (you can find this also in the section 2.1)
Line 81-82: not sure the meaning of this phrase; was the EPA production studied? how?
R: in the section 3.6 we reported the Fatty acids profile on the lipids extract from P. irregulare grown on EFJ medium and EFJ medium optimized after response surface (in terms of biomass productivity), with the relative lipids and EPA yield. We have not done an in-depth study on maximizing lipid productivity, but we have evaluated the differences between the various formulated mediums
Line 91: which is the composition of the V-8 Juice medium?
R: V-8 juice medium is tomato sauce based, with a supplementation of 3 g/L of CaCO3 , but in the first trials we found poor growth performances if compared to the YEP medium
Line 99: what is the modified YEP medium?
R: YEP medium is a standard growth medium for heterotrophic protist, it is based mainly on yeast extract and peptone with an addiction of glucose or sucrose. We used intensively this medium for the growth of the thraustochytrid Aurantiochytrium mangrovei
Line 115: what was the concentration of yeast extract in these culture experiments?
R: The concentration used was 2 g/L, as reported previously in the standard medium recipe.
Line 120: was glucose used as carbon source in these experiments? At what concentration?
R: Yes glucose was used as organic carbon, with a concentration of 30 g/L. We specified this in the text.
Line 141: what was the chemical composition of the food by-products and waste used? Although these are used as sources of C or N, they also contain sugars, carbohydrates and nitrogen (as proteins and peptides).
R: We agree with you, in fact by-products such as corn steep liquor can act as both a source of nitrogen and carbon, however we have made a macro-subdivision based on the main component present in the waste under consideration. Regarding the food waste composition, we specified this in the manuscript.
Line 232: in the experiment with lactose, was residual galactose detected?
R: in our study we did not evaluated the residual nutrients in the culture medium
Line 232: what was the effect of the C source on the EPA synthesis and lipid content of the biomass?
R: We have not evaluated the difference in EPA with various sources of organic carbon, but we checked the lipid content, and there were no significant differences between the various samples, so we did not report it in the paper.
Line 249: what was the effect of glucose concentration on the EPA synthesis and lipid content of the biomass?
R: in this case, we did not performed analysis on the EPA synthesis nor for the lipid content of the biomass grown with different glucose concentrations.
Line 253: the quality of the graphs should be improved.
R: we are sorry for this, during the submission we sent figures in 600 DPI so there was probably some problem with transposing the proof
Line 254: caption to the figure is not self explanatory; which was the carbon source used? What was the concentration? What was the concentration of the different N sources? In graph B, what was the N source?
R: we fixed the figure 3 caption and specified all the details.
Line 262-264: this explanation is not clear.
R: We reported a study from scientific literature which highlights the pathway in which P. irregulare metabolizes nitrate and ammonium from the extracellular environment for the production of glutamate, glutamine, proline, and arginine. We used this reference to prove the versatility of this microorganism, unlike S. cerevisiae and Y. lipolitica which cannot consume nitrate and nitrite as a nitrogen sources
Line 267: amino acids synthesis is an energy demanding process and microorganism save this energy using preformed amino acids. Besides amino acids are sources of both carbon and nitrogen.
R: we agree with that, in fact we tried also to report some studies which reports specific aminoacids transmembrane transporters for P. irregulare.
Line 271-274: this explanation is not clear.
R: as previously mentioned, we have tried to explain why Pythium irregulare is able to metabolize organic nitrogen faster than inorganic one
Line 280: which was the N source?
R: the N source used was yeast extract, we specified this in the manuscript
Line 287: N sources cannot be compared in terms of N content.
R: In order to compare various forms of nitrogen to be used, we formulated mediums that had a very similar nitrogen concentration. In the case of Pythium irregulare we have observed that the nitrogen requirement is very low in order to achieve a significant biomass growth.
Line 288: what was the effect of the N sources and concentration on the EPA synthesis and lipid content of the biomass?
R: we did not performed analysis on the EPA synthesis nor for the lipid content of the biomass grown with different nitrogen concentrations.
Line 306: a low temperature affects the kinetic of the process, and productivity instead of yield is decreased.
R: as you suggested, we fixed this sentence
Line 313: for the conditions used in this work, what was the effect of the incubation temperature on the EPA synthesis and lipid content of the biomass?
R: we evaluated the EPA yield at different temperatures, but as reported in the literature, even if the percentage on TFA increased, the yield decreased due to the lower biomass productivity. For that reason we did not considered this relevant for the research, but we can provide some data if required.
Line 325: for the conditions used in this work, what was the effect of salinity on the EPA synthesis and lipid content of the biomass?
R: we did not performed analysis on the EPA synthesis nor for the lipid content of the biomass grown with different saline concentrations as we only wanted to understand the ability of the biomass to grow even with high saline stress.
Line 350-351: which was the food by-product/waste that contained NaCl?
R: Cheese whey is enriched with NaCl during the cheese manufacturing, also molasses and corn steep liquor are by-product with a high mineral concentration that could affect the biomass growth
Line 357: which was the sugar composition of the juice?
R: The EFJ sugar composition was 141 ± 2 g L-1 of total sugars, of which 59% non reducing sugars (sucrose) and 41% reducing sugars (glucose and fructose)
Line 381: please check comas and symbols in the equation
R: We fixed this in the manuscript.
Line 395: please explain why the inhibition to sporulation would reduce growth performance.
R: a reduced sporulation rate affect directly the mycelium growth in some Pythium species, we specified this in the manuscript.
Line 406: if the objective of the study was the production of EPA then why EPA concentration was not used as the response for the optimization?
R: As we stated before, the main aim of our study was to evaluate the growth of P. irregulare using alternative food waste-based mediums. The lipid productivity was a "secondary" aim of the study
Line 442-444: please explain; it is not clear the relation of this text with the experimental results.
R: We specified this sentence better in the manuscript. In our case we wanted to explain that the biosynthesis of EPA in the pythium family is still an unclear topic that needs to be explored.
Line 452: which is the contribution of EFJ (used as source of sugars) and SBY (used as source of N) in terms of nitrogen and carbon, respectively?
R: in the optimized medium we supplemented 52 g L-1 of sugars from EFJ and 9.2 g L-1 of SBY. In terms of nitrogen this corresponds to 0.87 g L-1. We specified this in the manuscript.
Reviewer 4 Report
Paper titled “New biotechnological production of EPA by Pythium irregulare using alternative sustainable mediums obtained from food industry by-products and waste” by Russo et al report the use of Pythium irregulare as a potential source of eicosapentaenoic acid production using food industry by-products such as sugar cane molasses, spent brewery yeast, cheese whey and expired orange juice. The result shows that expired orange juice and spent brewery yeast after optimization, gave a biomass yield of 14.27 g/L, lipid yield of 2.23 g/L and 155 mg/L of eicosapentaenoic acid content.
The authors wrote the manuscript well. The research is novel and it is contributing significantly to the existing body of knowledge. Therefore I recommend the manuscript to be accepted for publication in Sustainability.
Author Response
Dear referee,
The authors wants to thank you for your time and considerations.
We are very glad that you liked the paper and that you found it interesting.
Best regards.
Round 2
Reviewer 3 Report
This second version of the manuscript has addressed most the comments raised in the review.
The authors have responded to all comments.
