Biotechnological Conversions of Mizithra Second Cheese Whey by Wild-Type Non-Conventional Yeast Strains: Production of Yeast Cell Biomass, Single-Cell Oil and Polysaccharides
, Dimitris Karayannis 1, Theofilos Massouras 2, Ioannis Politis 3 and Seraphim Papanikolaou 1,*Round 1
Reviewer 1 Report
Dear editor,
The submitted manuscript describes a strain and medium selection process for single cell oil fermentation bioprocess that aims to valorize waste cheese way as a carbon source. The manuscript may appeal to several scope items of the Applied Sciences, it is expertly conducted and worthy of publication in your journal. The strain selected in this study can be a starting point for optimization studies and has a potential to be scaled up in industrial level to utilize this valuable by-product.
General comments:
Please italicize all organism names. I think this was not intentional, but some formatting was lost probably during copying the content to the template. Please also check super- and sub-scripts and any other formatting issues. (e.g. CO2 in line 62, 160x106 and 45x106 in line 75, etc.)
I think the main objective is to provide lipids for the biofuel industry. How about its use in animal or human food? Are the strains used GRAS or safe enough to suggest human or animal consumption?
One room for improving this study would be the optimization of lipid production conditions, preferably at larger scale bioreactors. This can be a future study direction.
Lines 37-42: Please remove text provided in the article template.
Lines 64-70: By-product valorization has been discussed for some decades. However, why should we call it “meta-industry” now? SCO is a new topic but has been around for more than 10 years and I don’t know how it is “more innovative” than the already existing biomass valorization studies.
Line 91: (Mizithra, Anthotyro, Manouri): is it a reference? [26] has different names. Please cite properly.
Lines 493-500: Proposed bioprocess converts lactose to lipids, however, the current study does not report the organic load of the resulting waste after the lipids are extracted. Therefore, it is far reaching to imply that the proposed process will reduce the environmental impact significantly. Lactose is mostly removed but the fate of other organic compounds is largely unknown within the concept provided by this study.
Table 1: Please indicate the measure after plus-minus sign as (S.E.). Please also report “n” on the table so that the variance can be back-calculated.
Table 2: Line 723: “Material and Methods”: Please refer to the section with the section number.
Author Response
Important note: All points that have been eliminated are highlighted with red while all points that are added are highlighted with yellow.
Our responses concerning the reports follow.
REFEREE 1
We would like to express our thanks and appreciation for your comments concerning our work. Please see our responses:
1) “Please italicize all organism names. I think this was not intentional, but some formatting was lost probably during copying the content to the template. Please also check super- and sub-scripts and any other formatting issues. (e.g. CO2 in line 62, 160x106 and 45x106 in line 75, etc.)”
Response: Indeed, the formatting was lost during the copying to the template, thus we have tried to fixe everything according to our initial document and your comment.
2) “I think the main objective is to provide lipids for the biofuel industry. How about its use in animal or human food? Are the strains used GRAS or safe enough to suggest human or animal consumption?”
Response: We have added a paragraph the “Introduction” section dealing with this concept (please see also lines 120-130).
3) “One room for improving this study would be the optimization of lipid production conditions, preferably at larger scale bioreactors. This can be a future study direction.”
Response: Thank you very much. We agree with this point, it is a good proposal and we plan to implement it in near future (see also lines 613-617).
4) “Lines 37-42: Please remove text provided in the article template.”
Response: Thank you very much for noticing, this happened due to the transformation of our text into the template of the journal.
5) “Lines 64-70: By-product valorization has been discussed for some decades. However, why should we call it “meta-industry” now? SCO is a new topic but has been around for more than 10 years and I don’t know how it is “more innovative” than the already existing biomass valorization studies.” Response: Circular economy is a term that describes an economic model. The term “meta-industry” characterizes the industry itself, not the model of economy and describes the transition of the industry from classical industrial practice of product manufacture and waste disposal to an innovative next-generation industry, in which the by-product valorization would be an integral part of industrial practice and added-value metabolites such as SCO, organic acids, PHAs etc. could be produced. We would like to maintain in our text the term “meta-industry”, according to its translation from the ancient (and modern) Greek (see also lines 69-71).
5) “Line 91: (Mizithra, Anthotyro, Manouri): is it a reference? [26] has different names. Please cite properly.”
Response: It is not a reference, but the names of whey cheeses, widely produced in Greece.
6) “Lines 493-500: Proposed bioprocess converts lactose to lipids, however, the current study does not report the organic load of the resulting waste after the lipids are extracted. Therefore, it is far reaching to imply that the proposed process will reduce the environmental impact significantly. Lactose is mostly removed but the fate of other organic compounds is largely unknown within the concept provided by this study.”
Response: The initial concentrations of the compounds (Lactose, FAN, Protein, IP, TKN) in treated second cheese whey are presented in Table 1. For each individual microorganism, which was batch-cultivated in second CW, the consumption of each mentioned compound is mentioned in the text (Part 3.2.). Similarly, for the fed-batch experiment (Section 3.4.) the consumptions are also reported. Based on these data, the concentration of residues in wastewater can be easily calculated. Especially, in the wastewater resulting by fed-batch experiments, pollutant load was significantly reduced, because of the further assimilation of nitrogen sources. Certainly in the fed-batch experiment (see Fig. 3a) the remaining concentration of lactose at the end of the culture is significant but further incubation would reduce its concentration. Concerning the trials in the CW, with the exception of D. hansenii, all other microorganisms reduced significantly the polluting load of the waste. Comment concerning the detoxification / depollution potential of the employed strains are added in lines 419-424.
7) “Table 1: Please indicate the measure after plus-minus sign as (SE). Please also report “n” on the table so that the variance can be back-calculated.”
Response: As in the initial submission we have put the SD in this table. We consider that the SD value is satisfactory for simple tables like the Table 1.
8) “Table 2: Line 723: “Material and Methods”: Please refer to the section with the section number.” Response: Change was done.
REFEREE 2
Thank you very much for your valuable comments. Our responses are found in the next lines.
1) “Remove Lines 37-42, such mistakes should not take place, especially from the authors.”
Response: Done, thanks for noticing, we missed it by mistake.
2) “Lines 43-45: Provide relevant citations in this context for the stated fact of increased waste production.”
Response: You are right, it was our omission, the citation was added (see lines 620-621).
3) “Line 51: "being characterized as “"low added-value" ones” some waste are considered the main protein source, therefore they cannot be "considered low added-values" i.e. fish fodder, is mainly made either from fish waste or animal by products as the main protein source. Please provide more citations or reevaluate the statement.”
Response: We referred to the majority of solid or semi-solid agro-industrial wastes, of course there are exceptions, like the one you rightly mentioned (the word “waste” will be changed by the word “byproducts”). In order not to provoke confusions, we have omitted the phrase “"low added-value" ones” (see lines 51-52).
4) Line 85: “"that would further be used as food additives [16,17] have been proposed" Anhydrous cheese whey (CW) is actually being currently sold as supplement in the body building/fitness industry so is rather unclear what product is considered undesirable as waste. Please clarify and properly state the composition of second CW.”
Response: We have done the addition requested concerning the utilization of anhydrous CW as food supplement (see lines 89-90).
5) “Lines 129-130: "Finally, the strains with the codes NRRL Y- and NRRL YB- were kindly gifted by the NRRL Culture Collection (Peoria, Illinois, U.S.A.)" has to be moved to "Acknowledgements", this sentence has no place under material and methods.”
Response: These strains were not gifted, they were provided. We consider that the origin of the strains should be maintained in the section “Materials and Methods”.
5) “Line 190: "sterilized in autoclave" state the temperature and duration.”
Response: Done, see line 208.
6) “Line 191: "cultures were incubated, as described above" be that 30 C 180 rpm, was the pH adjusted in this case as well?”
Response: Yes, at 30 oC and 180 rpm, with pH adjustment, that is why we have said “as described above”. In other case, we would describe in detail.
7) “Lines 215-216: "Residual lactose (S, g/L), total dry weight (TDW) of biomass (X, g/L) and cellular lipids (L, g/L) were determined" How?”
Response: It is described below, at the corresponding section (see lines 234-235).
8) “Lines 216-219: "pH, yield or substrate to biomass coefficient (YX/S, expressed in g/g), substrate to lipids coefficient (YL/S, expressed in g/g), dry biomass productivity (PX, expressed in g/L/h), lipid productivity (PL, expressed in g/L/h), and lipid content in dry biomass (KL/X, expressed in g/g) were calculated." How?”
Response: All parameters were calculated according to Papanikolaou et al., (2001). See also additions in lines 235-241.
9) “Lines 220-224: again, methods are not stated”.
Response: Everything is clearly indicated in lines 247-291.
10) “Lines 215-224 require rephrasing as in stating the protocols involved in a clear step by step manner. The purpose of a scientific article is also to be reproducible.”
Response: Please see lines 236-241. In any case we consider that we present clearly in the next paragraphs, (lines 247-291) all techniques.
11) “HPLC data acquisition was performed at what wavelength? What was the injection volume? Isocratic or gradient elution?”
Response: The sugars were detected by Refractive Index Detector, Waters 2410 at 880 nm. The injection volume was 20 μL and the elution was isocratic. Everything is mentioned in the text (see lines 262-265.
11) “"The results were quantified, corresponding to standards." present the standard.”
Response: The laboratory has standard curves of the various commercial sugars, through which the eluates were identified and quantified. We do not consider that a calibration curve adds something the important in this paper.
12) “Line 270: " as described above" it's not described, please clearly describe the method and the wavelength as well as the reason why”.
Response: We believe that the procedure and the usage of the HPLC apparatus are, now, fully described (see lines 258-265).
12) “Results. Where are the HPLC chromatograms?”
Response: The current study consists of several experiments, with many HPLC analyzes for each kinetics performed. We believe that listing the chromatograms in the text does not add importantly to the essence of the study. For your information, we attach, to this file, an indicative chromatogram, from which and based on the corresponding standard curve, for example for lactose, the sugar concentration was determined.
13) Conclusions have been integrated in Discussions? Please separate.
Response: Done.
Author Response File: 
Author Response.docx
Reviewer 2 Report
Vasilakis et al. evaluated seven non-conventional yeast strains with the agro-industrial waste cheese-whey lactose as a sole carbon source for biomass production with a latter scope of obtaining single-cell oils.
Remove Lines 37-42, such mistakes should not take place, especially from the authors.
Lines 43-45: Provide relevant citations in this context for the stated fact of increased waste production.
Line 51: "being characterized as “low added-value” ones" some waste are considered the main protein source, therefore they can not be "considered low added-values" i.e. fish fodder, is mainly made either from fish waste or animal by products as the main protein source. Please provide more citations or reevaluate the statement.
Line 85: "that would further be used as food additives [16,17] have been proposed" Anhydrous cheese whey (CW) is actually being currently sold as supplement in the body building/fitness industry so is rather unclear what product is considered undesirable as waste. Please clarify and properly state the composition of second CW.
Lines 129-130: "Finally, the strains with the codes NRRL Y- and NRRL YB- were kindly gifted by the NRRL Culture Collection (Peoria, Illinois, U.S.A.)" has to be moved to "Acknowledgements", this sentence has no place under material and methods.
Line 190: "sterilized in autoclave" state the temperature and duration.
Line 191: "cultures were incubated, as described above" be that 30 C 180 rpm, was the pH adjusted in this case as well?
Lines 215-216: "Residual lactose (S, g/L), total dry weight (TDW) of biomass (X, g/L) and cellular lipids (L, g/L) were determined" How?
Lines 216-219: "pH, yield or substrate to biomass coefficient (YX/S, expressed in g/g), substrate to lipids coefficient (YL/S, expressed in g/g), dry biomass productivity (PX, expressed in g/L/h), lipid productivity (PL, expressed in g/L/h), and lipid content in dry biomass (KL/X, expressed in g/g) were calculated." How?
Lines 220-224: again, methods are not stated
Lines 215-224 require rephrasing as in stating the protocols involved in a clear step by step manner. The purpose of an scientific article is also to be reproducible.
HPLC data acquisition was performed at what wavelength? What was the injection volume? Isocratic or gradient elution?
"The results were quantified, corresponding to standards." present the standard.
Line 270: " as described above" it's not described, please clearly describe the method and the wavelength as well as the reason why
Results
Where are the HPLC chromatograms?
Conclusions have been integrated in Discussions? Please separate
Author Response
Important note: All points that have been eliminated are highlighted with red while all points that are added are highlighted with yellow.
Our responses concerning the reports follow.
REFEREE 1
We would like to express our thanks and appreciation for your comments concerning our work. Please see our responses:
1) “Please italicize all organism names. I think this was not intentional, but some formatting was lost probably during copying the content to the template. Please also check super- and sub-scripts and any other formatting issues. (e.g. CO2 in line 62, 160x106 and 45x106 in line 75, etc.)”
Response: Indeed, the formatting was lost during the copying to the template, thus we have tried to fixe everything according to our initial document and your comment.
2) “I think the main objective is to provide lipids for the biofuel industry. How about its use in animal or human food? Are the strains used GRAS or safe enough to suggest human or animal consumption?”
Response: We have added a paragraph the “Introduction” section dealing with this concept (please see also lines 120-130).
3) “One room for improving this study would be the optimization of lipid production conditions, preferably at larger scale bioreactors. This can be a future study direction.”
Response: Thank you very much. We agree with this point, it is a good proposal and we plan to implement it in near future (see also lines 613-617).
4) “Lines 37-42: Please remove text provided in the article template.”
Response: Thank you very much for noticing, this happened due to the transformation of our text into the template of the journal.
5) “Lines 64-70: By-product valorization has been discussed for some decades. However, why should we call it “meta-industry” now? SCO is a new topic but has been around for more than 10 years and I don’t know how it is “more innovative” than the already existing biomass valorization studies.” Response: Circular economy is a term that describes an economic model. The term “meta-industry” characterizes the industry itself, not the model of economy and describes the transition of the industry from classical industrial practice of product manufacture and waste disposal to an innovative next-generation industry, in which the by-product valorization would be an integral part of industrial practice and added-value metabolites such as SCO, organic acids, PHAs etc. could be produced. We would like to maintain in our text the term “meta-industry”, according to its translation from the ancient (and modern) Greek (see also lines 69-71).
5) “Line 91: (Mizithra, Anthotyro, Manouri): is it a reference? [26] has different names. Please cite properly.”
Response: It is not a reference, but the names of whey cheeses, widely produced in Greece.
6) “Lines 493-500: Proposed bioprocess converts lactose to lipids, however, the current study does not report the organic load of the resulting waste after the lipids are extracted. Therefore, it is far reaching to imply that the proposed process will reduce the environmental impact significantly. Lactose is mostly removed but the fate of other organic compounds is largely unknown within the concept provided by this study.”
Response: The initial concentrations of the compounds (Lactose, FAN, Protein, IP, TKN) in treated second cheese whey are presented in Table 1. For each individual microorganism, which was batch-cultivated in second CW, the consumption of each mentioned compound is mentioned in the text (Part 3.2.). Similarly, for the fed-batch experiment (Section 3.4.) the consumptions are also reported. Based on these data, the concentration of residues in wastewater can be easily calculated. Especially, in the wastewater resulting by fed-batch experiments, pollutant load was significantly reduced, because of the further assimilation of nitrogen sources. Certainly in the fed-batch experiment (see Fig. 3a) the remaining concentration of lactose at the end of the culture is significant but further incubation would reduce its concentration. Concerning the trials in the CW, with the exception of D. hansenii, all other microorganisms reduced significantly the polluting load of the waste. Comment concerning the detoxification / depollution potential of the employed strains are added in lines 419-424.
7) “Table 1: Please indicate the measure after plus-minus sign as (SE). Please also report “n” on the table so that the variance can be back-calculated.”
Response: As in the initial submission we have put the SD in this table. We consider that the SD value is satisfactory for simple tables like the Table 1.
8) “Table 2: Line 723: “Material and Methods”: Please refer to the section with the section number.” Response: Change was done.
REFEREE 2
Thank you very much for your valuable comments. Our responses are found in the next lines.
1) “Remove Lines 37-42, such mistakes should not take place, especially from the authors.”
Response: Done, thanks for noticing, we missed it by mistake.
2) “Lines 43-45: Provide relevant citations in this context for the stated fact of increased waste production.”
Response: You are right, it was our omission, the citation was added (see lines 620-621).
3) “Line 51: "being characterized as “"low added-value" ones” some waste are considered the main protein source, therefore they cannot be "considered low added-values" i.e. fish fodder, is mainly made either from fish waste or animal by products as the main protein source. Please provide more citations or reevaluate the statement.”
Response: We referred to the majority of solid or semi-solid agro-industrial wastes, of course there are exceptions, like the one you rightly mentioned (the word “waste” will be changed by the word “byproducts”). In order not to provoke confusions, we have omitted the phrase “"low added-value" ones” (see lines 51-52).
4) Line 85: “"that would further be used as food additives [16,17] have been proposed" Anhydrous cheese whey (CW) is actually being currently sold as supplement in the body building/fitness industry so is rather unclear what product is considered undesirable as waste. Please clarify and properly state the composition of second CW.”
Response: We have done the addition requested concerning the utilization of anhydrous CW as food supplement (see lines 89-90).
5) “Lines 129-130: "Finally, the strains with the codes NRRL Y- and NRRL YB- were kindly gifted by the NRRL Culture Collection (Peoria, Illinois, U.S.A.)" has to be moved to "Acknowledgements", this sentence has no place under material and methods.”
Response: These strains were not gifted, they were provided. We consider that the origin of the strains should be maintained in the section “Materials and Methods”.
5) “Line 190: "sterilized in autoclave" state the temperature and duration.”
Response: Done, see line 208.
6) “Line 191: "cultures were incubated, as described above" be that 30 C 180 rpm, was the pH adjusted in this case as well?”
Response: Yes, at 30 oC and 180 rpm, with pH adjustment, that is why we have said “as described above”. In other case, we would describe in detail.
7) “Lines 215-216: "Residual lactose (S, g/L), total dry weight (TDW) of biomass (X, g/L) and cellular lipids (L, g/L) were determined" How?”
Response: It is described below, at the corresponding section (see lines 234-235).
8) “Lines 216-219: "pH, yield or substrate to biomass coefficient (YX/S, expressed in g/g), substrate to lipids coefficient (YL/S, expressed in g/g), dry biomass productivity (PX, expressed in g/L/h), lipid productivity (PL, expressed in g/L/h), and lipid content in dry biomass (KL/X, expressed in g/g) were calculated." How?”
Response: All parameters were calculated according to Papanikolaou et al., (2001). See also additions in lines 235-241.
9) “Lines 220-224: again, methods are not stated”.
Response: Everything is clearly indicated in lines 247-291.
10) “Lines 215-224 require rephrasing as in stating the protocols involved in a clear step by step manner. The purpose of a scientific article is also to be reproducible.”
Response: Please see lines 236-241. In any case we consider that we present clearly in the next paragraphs, (lines 247-291) all techniques.
11) “HPLC data acquisition was performed at what wavelength? What was the injection volume? Isocratic or gradient elution?”
Response: The sugars were detected by Refractive Index Detector, Waters 2410 at 880 nm. The injection volume was 20 μL and the elution was isocratic. Everything is mentioned in the text (see lines 262-265.
11) “"The results were quantified, corresponding to standards." present the standard.”
Response: The laboratory has standard curves of the various commercial sugars, through which the eluates were identified and quantified. We do not consider that a calibration curve adds something the important in this paper.
12) “Line 270: " as described above" it's not described, please clearly describe the method and the wavelength as well as the reason why”.
Response: We believe that the procedure and the usage of the HPLC apparatus are, now, fully described (see lines 258-265).
12) “Results. Where are the HPLC chromatograms?”
Response: The current study consists of several experiments, with many HPLC analyzes for each kinetics performed. We believe that listing the chromatograms in the text does not add importantly to the essence of the study. For your information, we attach, to this file, an indicative chromatogram, from which and based on the corresponding standard curve, for example for lactose, the sugar concentration was determined.
13) Conclusions have been integrated in Discussions? Please separate.
Response: Done.
Author Response File: Author Response.docx
Reviewer 3 Report
The manuscript can be accepted in its present form.
Author Response
Important note: All points that have been eliminated are highlighted with red while all points that are added are highlighted with yellow.
Our responses concerning the reports follow.
REFEREE 1
We would like to express our thanks and appreciation for your comments concerning our work. Please see our responses:
1) “Please italicize all organism names. I think this was not intentional, but some formatting was lost probably during copying the content to the template. Please also check super- and sub-scripts and any other formatting issues. (e.g. CO2 in line 62, 160x106 and 45x106 in line 75, etc.)”
Response: Indeed, the formatting was lost during the copying to the template, thus we have tried to fixe everything according to our initial document and your comment.
2) “I think the main objective is to provide lipids for the biofuel industry. How about its use in animal or human food? Are the strains used GRAS or safe enough to suggest human or animal consumption?”
Response: We have added a paragraph the “Introduction” section dealing with this concept (please see also lines 120-130).
3) “One room for improving this study would be the optimization of lipid production conditions, preferably at larger scale bioreactors. This can be a future study direction.”
Response: Thank you very much. We agree with this point, it is a good proposal and we plan to implement it in near future (see also lines 613-617).
4) “Lines 37-42: Please remove text provided in the article template.”
Response: Thank you very much for noticing, this happened due to the transformation of our text into the template of the journal.
5) “Lines 64-70: By-product valorization has been discussed for some decades. However, why should we call it “meta-industry” now? SCO is a new topic but has been around for more than 10 years and I don’t know how it is “more innovative” than the already existing biomass valorization studies.” Response: Circular economy is a term that describes an economic model. The term “meta-industry” characterizes the industry itself, not the model of economy and describes the transition of the industry from classical industrial practice of product manufacture and waste disposal to an innovative next-generation industry, in which the by-product valorization would be an integral part of industrial practice and added-value metabolites such as SCO, organic acids, PHAs etc. could be produced. We would like to maintain in our text the term “meta-industry”, according to its translation from the ancient (and modern) Greek (see also lines 69-71).
5) “Line 91: (Mizithra, Anthotyro, Manouri): is it a reference? [26] has different names. Please cite properly.”
Response: It is not a reference, but the names of whey cheeses, widely produced in Greece.
6) “Lines 493-500: Proposed bioprocess converts lactose to lipids, however, the current study does not report the organic load of the resulting waste after the lipids are extracted. Therefore, it is far reaching to imply that the proposed process will reduce the environmental impact significantly. Lactose is mostly removed but the fate of other organic compounds is largely unknown within the concept provided by this study.”
Response: The initial concentrations of the compounds (Lactose, FAN, Protein, IP, TKN) in treated second cheese whey are presented in Table 1. For each individual microorganism, which was batch-cultivated in second CW, the consumption of each mentioned compound is mentioned in the text (Part 3.2.). Similarly, for the fed-batch experiment (Section 3.4.) the consumptions are also reported. Based on these data, the concentration of residues in wastewater can be easily calculated. Especially, in the wastewater resulting by fed-batch experiments, pollutant load was significantly reduced, because of the further assimilation of nitrogen sources. Certainly in the fed-batch experiment (see Fig. 3a) the remaining concentration of lactose at the end of the culture is significant but further incubation would reduce its concentration. Concerning the trials in the CW, with the exception of D. hansenii, all other microorganisms reduced significantly the polluting load of the waste. Comment concerning the detoxification / depollution potential of the employed strains are added in lines 419-424.
7) “Table 1: Please indicate the measure after plus-minus sign as (SE). Please also report “n” on the table so that the variance can be back-calculated.”
Response: As in the initial submission we have put the SD in this table. We consider that the SD value is satisfactory for simple tables like the Table 1.
8) “Table 2: Line 723: “Material and Methods”: Please refer to the section with the section number.” Response: Change was done.
REFEREE 2
Thank you very much for your valuable comments. Our responses are found in the next lines.
1) “Remove Lines 37-42, such mistakes should not take place, especially from the authors.”
Response: Done, thanks for noticing, we missed it by mistake.
2) “Lines 43-45: Provide relevant citations in this context for the stated fact of increased waste production.”
Response: You are right, it was our omission, the citation was added (see lines 620-621).
3) “Line 51: "being characterized as “"low added-value" ones” some waste are considered the main protein source, therefore they cannot be "considered low added-values" i.e. fish fodder, is mainly made either from fish waste or animal by products as the main protein source. Please provide more citations or reevaluate the statement.”
Response: We referred to the majority of solid or semi-solid agro-industrial wastes, of course there are exceptions, like the one you rightly mentioned (the word “waste” will be changed by the word “byproducts”). In order not to provoke confusions, we have omitted the phrase “"low added-value" ones” (see lines 51-52).
4) Line 85: “"that would further be used as food additives [16,17] have been proposed" Anhydrous cheese whey (CW) is actually being currently sold as supplement in the body building/fitness industry so is rather unclear what product is considered undesirable as waste. Please clarify and properly state the composition of second CW.”
Response: We have done the addition requested concerning the utilization of anhydrous CW as food supplement (see lines 89-90).
5) “Lines 129-130: "Finally, the strains with the codes NRRL Y- and NRRL YB- were kindly gifted by the NRRL Culture Collection (Peoria, Illinois, U.S.A.)" has to be moved to "Acknowledgements", this sentence has no place under material and methods.”
Response: These strains were not gifted, they were provided. We consider that the origin of the strains should be maintained in the section “Materials and Methods”.
5) “Line 190: "sterilized in autoclave" state the temperature and duration.”
Response: Done, see line 208.
6) “Line 191: "cultures were incubated, as described above" be that 30 C 180 rpm, was the pH adjusted in this case as well?”
Response: Yes, at 30 oC and 180 rpm, with pH adjustment, that is why we have said “as described above”. In other case, we would describe in detail.
7) “Lines 215-216: "Residual lactose (S, g/L), total dry weight (TDW) of biomass (X, g/L) and cellular lipids (L, g/L) were determined" How?”
Response: It is described below, at the corresponding section (see lines 234-235).
8) “Lines 216-219: "pH, yield or substrate to biomass coefficient (YX/S, expressed in g/g), substrate to lipids coefficient (YL/S, expressed in g/g), dry biomass productivity (PX, expressed in g/L/h), lipid productivity (PL, expressed in g/L/h), and lipid content in dry biomass (KL/X, expressed in g/g) were calculated." How?”
Response: All parameters were calculated according to Papanikolaou et al., (2001). See also additions in lines 235-241.
9) “Lines 220-224: again, methods are not stated”.
Response: Everything is clearly indicated in lines 247-291.
10) “Lines 215-224 require rephrasing as in stating the protocols involved in a clear step by step manner. The purpose of a scientific article is also to be reproducible.”
Response: Please see lines 236-241. In any case we consider that we present clearly in the next paragraphs, (lines 247-291) all techniques.
11) “HPLC data acquisition was performed at what wavelength? What was the injection volume? Isocratic or gradient elution?”
Response: The sugars were detected by Refractive Index Detector, Waters 2410 at 880 nm. The injection volume was 20 μL and the elution was isocratic. Everything is mentioned in the text (see lines 262-265.
11) “"The results were quantified, corresponding to standards." present the standard.”
Response: The laboratory has standard curves of the various commercial sugars, through which the eluates were identified and quantified. We do not consider that a calibration curve adds something the important in this paper.
12) “Line 270: " as described above" it's not described, please clearly describe the method and the wavelength as well as the reason why”.
Response: We believe that the procedure and the usage of the HPLC apparatus are, now, fully described (see lines 258-265).
12) “Results. Where are the HPLC chromatograms?”
Response: The current study consists of several experiments, with many HPLC analyzes for each kinetics performed. We believe that listing the chromatograms in the text does not add importantly to the essence of the study. For your information, we attach, to this file, an indicative chromatogram, from which and based on the corresponding standard curve, for example for lactose, the sugar concentration was determined.
13) Conclusions have been integrated in Discussions? Please separate.
Response: Done.
Author Response File: Author Response.docx
Round 2
Reviewer 2 Report
The authors have improved their manuscript considerable.
