Lipid and Carotenoid Production by Rhodotorula glutinis with a Combined Cultivation Mode of Nitrogen, Sulfur, and Aluminium Stress
Round 1
Reviewer 1 Report
The paper aims at defining cultivation conditions maximizing the production of the carotenoid torulene. The molecular basis of torulene biosynthesis are yet to be clarified, therefore adjustments on the nutrients ratio during cultivation is at present time, the quickest approach to detect changes in the carotenoid's production. Both adjustments of the culture conditions and stress exposure were tested to assess changes in the pigments' abundance profile as compared to the production of lipids. The effects of different carbon and nitrogen sources as well as aluminium sulfate as stress factor were assessed by measurement of dry biomass weight, lipid (Nile red staining and sulfo-phospho vanillin method) and carotenoid content (HPLC). As a result, the authors detect the highest yield of carotenoids in relation to a combination of C/N 146, C/S 120 and ammonium sulfate as N source. Moreover, they report an enhancement in the synthesis of total pigment, thus of toluene as well, after supply of aluminium sulfate, this confirming the role of metal stress on carotenoids' accumulation.
The general idea at the base of the manuscript is valuable as it aims at the tuning of yeasts' cultivation conditions to improve production of carotenoid compounds, specifically torulene, with possibly broad biotechnological applications. Nevertheless, the writing style substantially hinders the reading and the comprehension of the text to the detriment of the research work. An extensive editing of both English language and style is therefore highly suggested. Additionally, the results are not well presented and the bar graphs (i.e. Figure 2) would probably need to be revised (size and choice of colors) as it would make it easier to extrapolate the results. The introduction could be extended to include additional background information about the yeast strain used in the study, the cultivation preferences and the role of metal stress, which is only briefly mentioned. In a similar fashion, the discussion section should undergo amendments in order to improve clarity.
Overall, it is an interesting study, and should be considered for publication in Applied Sciences only once the language and style issue has been resolved.
Author Response
Comment 1: The paper aims at defining cultivation conditions maximizing the production of the carotenoid torulene. The molecular basis of torulene biosynthesis are yet to be clarified, therefore adjustments on the nutrients ratio during cultivation is at present time, the quickest approach to detect changes in the carotenoid's production. Both adjustments of the culture conditions and stress exposure were tested to assess changes in the pigments' abundance profile as compared to the production of lipids. The effects of different carbon and nitrogen sources as well as aluminium sulfate as stress factor were assessed by measurement of dry biomass weight, lipid (Nile red staining and sulfo-phospho vanillin method) and carotenoid content (HPLC). As a result, the authors detect the highest yield of carotenoids in relation to a combination of C/N 146, C/S 120 and ammonium sulfate as N source. Moreover, they report an enhancement in the synthesis of total pigment, thus of toluene as well, after supply of aluminium sulfate, this confirming the role of metal stress on carotenoids' accumulation. The general idea at the base of the manuscript is valuable as it aims at the tuning of yeasts' cultivation conditions to improve production of carotenoid compounds, specifically torulene, with possibly broad biotechnological applications. Nevertheless, the writing style substantially hinders the reading and the comprehension of the text to the detriment of the research work. An extensive editing of both English language and style is therefore highly suggested.
Answer: Thanks for reviewer’s advice. The manuscript was revised. Changes are highlighted with red color in the revised manuscript.
Comment 2: The results are not well presented and the bar graphs (i.e. Figure 2) would probably need to be revised (size and choice of colors) as it would make it easier to extrapolate the results.
Answer: We appreciate the reviewer’s constructive suggestion. The result section was rewritten to clarify the most important finding of this study. And figure 2, figure 4, and figure 5 were changed to table 3, 5 and 6, respectively.
Comment 3: The introduction could be extended to include additional background information about the yeast strain used in the study, the cultivation preferences and the role of metal stress.
Answer: We appreciate the reviewer’s constructive suggestion. In introduction section, we supplemented paragraphs about the microbe producing carotenoid and focused on our studied strain, cultivation condition and effect of metal on living organisms. Changes are highlighted with red color in the revised manuscript.
Comment 4: Discussion section should undergo amendments in order to improve clarity.
Answer: Thanks for reviewer’s advice. Discussion section was revised to improve clarity. Changes are highlighted with red color in the revised manuscript.
Reviewer 2 Report
Generally, in my opinion the manuscript contain interesting information and should be interest to the readers of the Applied Sciences. It is better if the authors consider the following mentioned remarks and further improve the manuscript before submitting the final version.
Some drawbacks are listed below in the order of the manuscript:
After reading the whole manuscript, I think that its current title does not reflect the overall idea of work. Firstly, in addition to the biosynthesis of carotenoids, lipid biosynthesis is also an important element of the manuscript. Secondly, why this is POTENCIAL torulene accumulation? I leave my comment to be considered by the Authors and Editor of the journal.
Line 35-37 - current data from 2017 are available,
Line 45 – I do not know olic acid,
Line 48 – torularhodine not torularhodene
Line 54 – 55 “… safe food additives shown by toxicity studies in rats and inhibiting cells growth of prostate tumours” – I don’t understand the idea of these sentence, please rewrite it
Line 70 – The authors have given in acknowledgment the information from whom they obtained the test strain and it should be there, not in the methodology. Please explain the origin of the strain, the name of the collection or university / institute, in other articles is R. glutinis CGMCC 2.703 (is the same strain?)
Generally comment to methodology section: details of the equipment should be given. Testing methods should be described in detail or specific references should be provided.
Line 102 – 103 : it should be …a new fermentation medium WAS prepared…, next:
Different Al2(SO4)3 concentrations WERE added to…
Line 107-108 - the cells WERE washed twice…
Subchapters 2.4. and 2.5 are desribed too general, please provide details
Line 124 – 126 – repeating the same sentence twice
Why different wavelengths were used for spectrophotometric analysis (485 nm) and HPLC (501 nm)
Line 156 – literature or data not shown?
Line 184 – Do the authors know what it can be exactly a precursor?
Figure 5 is very difficult to read it, I suggests to replace them at the table
Line 265 – 266 Do the authors suspect what this enzyme may be?
Line 338 – torulene is a better antioxidant than what?
Line 380 – reference to table 3 but it is not in my version of the manuscript
Did the authors wonder why after the addition of aluminum sulphate the process of torularhodine biosynthesis was inhibited?
The list of literature should be improved, there are many mistakes in it.
Author Response
Comment 1: Overall, it is an interesting study, and should be considered for publication in Applied Sciences only once the language and style issue has been resolved. Generally, in my opinion the manuscript contain interesting information and should be interest to the readers of the Applied Sciences. It is better if the authors consider the following mentioned remarks and further improve the manuscript before submitting the final version.
Answer: Thanks for reviewer’s advice. The manuscript was revised. Changes are highlighted with red color in the revised manuscript.
Comment 2: After reading the whole manuscript, I think that its current title does not reflect the overall idea of work. Firstly, in addition to the biosynthesis of carotenoids, lipid biosynthesis is also an important element of the manuscript. Secondly, why this is POTENCIAL torulene accumulation? I leave my comment to be considered by the Authors and Editor of the journal.
Answer: We appreciate the reviewer’s constructive suggestion. The title was changed with: Lipid and carotenoid production by Rhodotorula glutinis with combined cultivation mode of nitrogen, sulfur and aluminium stress.
Comment 3
Some drawbacks are listed below in the order of the manuscript:
Comment 3.1 Line 35-37 - current data from 2017 are available,
Answer: Thanks for reviewer’s advice. We replaced this paragraph with the current data line 34, 35 in revised manuscript.
.
Comment 3.2 Line 45 – I do not know olic acid,
Answer: We are sorry for careless. It should be Oleic acid.
Comment 3.3 Line 48 – torularhodine not torularhodene
Answer: We corrected the spelling mistake of torularhodene with torularhodine throughout whole manuscript.
Comment 3.4 Line 54 – 55 “… safe food additives shown by toxicity studies in rats and inhibiting cells growth of prostate tumours” – I don’t understand the idea of these sentence, please rewrite it
Answer: Thanks for reviewer’s advice. We changed the paragraph as: Carotenoid torulene has 13 double bonds, one β-ionone and a longer polyene chain than β-carotene. Torulene exhibits properties of provitamin A and anti-prostate cancer activity and safe food additives. Line 51-53 in revised manuscript.
Comment 3.5 Line 70 – The authors have given in acknowledgment the information from whom they obtained the test strain and it should be there, not in the methodology. Please explain the origin of the strain, the name of the collection or university / institute, in other articles is R. glutinis CGMCC 2.703 (is the same strain?)
Answer: Thanks for reviewer’s advice. The origin of the yeast strain was revised in materials and methods section, line 85 in revised manuscript. The information of donator was added in acknowledgement section. Yes, the used strain is the same with R. glutinis CGMCC 2.703
Comment 3.6 Generally comment to methodology section: details of the equipment should be given. Testing methods should be described in detail or specific references should be provided.
Answer: Thanks for reviewer’s advice. We described all the method in detail except DNS method.
Comment 3.7 Line 102 – 103: it should be …a new fermentation medium WAS prepared…, next: Different Al2(SO4)3 concentrations WERE added to…
Answer: Thanks for reviewer’s advice. The grammar was corrected at line 118 in revised manuscript, and throughout the manuscript.
Comment 3.8 Line 107-108 - the cells WERE washed twice…
Answer: Thanks for reviewer’s advice. The repeat sentence was removed. And the grammar was corrected at line 121-122.
Comment 3.9 Subchapters 2.4. and 2.5 are desribed too general, please provide details
Answer: Thanks for reviewer’s advice. Subchapters were revised and detailed methods were added.
Comment 3.10 Line 124 – 126 – repeating the same sentence twice
Answer: Thanks for reviewer’s advice. The repeat sentence was removed.
Comment 3.11 Why different wavelengths were used for spectrophotometric analysis (485 nm) and HPLC (501 nm)
Answer: We appreciate the reviewer’s constructive suggestion. In spectrophotometric analysis, wavelength was used firstly. On HPLC detection, we optimized detected wavelength with 450, 470, 485 and 501 nm, 501 giving best result regarding separation and quantification. So, 501 nm was used for HPLC.
Comment 3.12 Line 156 – literature or data not shown?
Answer: Thanks for reviewer’s advice. We added data not shown and documented it in the literature section.
Comment 3.13 Line 184 – Do the authors know what it can be exactly a precursor?
Answer: Thanks for reviewer’s advice. We added acetyl CoA at line 216 in revised manuscript, the precursor of lipogenesis and carotenogenesis.
Comment 3.14 Figure 5 is very difficult to read it, I suggests to replace them at the table
Answer: We appreciate the reviewer’s constructive suggestion. We replaced figure 5 with table 6, also replaced figure 2 and 4 with table 3 and 5, respectively.
Comment 3.15 Line 265 – 266 Do the authors suspect what this enzyme may be?
Answer: Thanks for reviewer’s advice. We revised the expression as: This result indicated the reverse effect of aluminium sulfate on the lipogenesis pathway. Transcriptome analysis would be done, and the mechanism of effect of aluminium sulfate on lipid production could be clarified further.
Comment 3.16 Line 338 – torulene is a better antioxidant than what?
Answer: Thanks for reviewer’s advice. We revised expression as: torulene as better antioxidant than β-Carotene and γ-carotene-, and added reference at line 430 in revised manuscript.
Comment 3.17 Line 380 – reference to table 3 but it is not in my version of the manuscript
Answer: We are sorry for carless. We added Table 7.
Comment 3.18 Did the authors wonder why after the addition of aluminum sulphate the process of torularhodine biosynthesis was inhibited?
Answer: We appreciate the reviewer’s constructive suggestion. Generally, the studied strain is not good producer of torularhodine, and it was not detected also under other culture conditions not only with aluminum sulfate. Based on transcriptome analysis in future, effect of aluminium sulfate on torularhodine biosynthesis would be clarified.
Reviewer 3 Report
The paper is under the scope of the journal , and the findings are novel :The paper needs minor revisions on the following points:
The paper is lacking a hypothesis. Please correct.
Introduction part is inadequate and was written disorderly. Some of the important literature material that is important to the reader is missing. The clarity and coherence of expression and writing should be strengthened. What is the aim of the study, indicate clearly. Experimental section is documented unsatisfactorily.
The Methods is too short, please expand and not give a reference only.
Figures are too small, please correct for larger sizes. I do not see any differences.
Discussion: It is recommended improve writing.
Popraw dyskusje wyników i wstęp w artykule opisując dane:
(2016) Rhodotorula glutinis—potential source of lipids, carotenoids, and enzymes for use in industries. Applied Microbiology and Biotechnology, 100(14), 6103-6117.
Unfortunately, this is a purely descriptive article and does not provide any mechanistic insight.
We write the names of microorganisms in italics. Improve throughout the article.
Where is the statistical analysis of the results carried out? In this case, the results are unreliable and the article will be rejected. Please do it because it is very important.
Correct the references because they are badly prepared: Example:10, 13, 19, 33
Conclusion is very weak. Please expand and describe the most important results of the work. To interest the industrial reader in the use of industry results.
Author Response
Comment 1: The paper is under the scope of the journal, and the findings are novel: The paper needs minor revisions on the following points: The paper is lacking a hypothesis. Please correct
Answer: Thanks for reviewer’s advice. The manuscript was revised, and the hypothesis and aim of the paper were added according reviewer’s suggestion.
Comment 2: Introduction part is inadequate and was written disorderly. Some of the important literature material that is important to the reader is missing. The clarity and coherence of expression and writing should be strengthened. What is the aim of the study, indicate clearly.
Answer: Thanks for reviewer’s advice. We are sorry for expressing ambiguously. We re-wrote the introduction section.
Comment 3: The Methods is too short, please expand and not give a reference only.
Answer: Thanks for reviewer’s advice. Experimental section was revised and expanded.
Comment 4: Figures are too small, please correct for larger sizes. I do not see any differences.
Answer: Thanks for reviewer’s advice. All figures were revised and replaced with new ones.
Comment 5 Discussion: It is recommended improve writing.
Answer: Thanks for reviewer’s advice. Discussion of the manuscript was revised and polished. Changes are highlighted with red color in the revised manuscript.
Comment 6 Popraw dyskusje wyników i wstęp w artykule opisując dane:
(2016) Rhodotorula glutinis—potential source of lipids, carotenoids, and enzymes for use in industries. Applied Microbiology and Biotechnology, 100(14), 6103-6117.
Unfortunately, this is a purely descriptive article and does not provide any mechanistic insight. We write the names of microorganisms in italics. Improve throughout the article.
Where is the statistical analysis of the results carried out? In this case, the results are unreliable and the article will be rejected. Please do it because it is very important.
Answer: Thanks for reviewer’s advice. All the organism names are now in italic. Statistical analysis section was added, and statistical significant difference of data in figures and tables was carried out.
Comment 7 Correct the references because they are badly prepared: Example:10, 13, 19, 33
Answer: Thanks for reviewer’s advice. We are sorry for careless. We revised the reference one by one according to the journal format.
Comment 8 Conclusion is very weak. Please expand and describe the most important results of the work. To interest the industrial reader in the use of industry results.
Answer: Thanks for reviewer’s advice. We re-wrote the conclusion.
Round 2
Reviewer 3 Report
The authors have not improved their admission again. There is not reference to the latest research.
The authors can not find a link, draw conclusions because they do not read the latest scientific reports.
Introduction, methodology and discussion of results should be a kind of correct history that smoothly goes to the conclusion.
In the vale, it is worth describing the possibilities of producing lipids by the Rhodotorula strain. This information is missing. There are very few of them
In the article, not only should new sentences be added in red but also conclusions should be drawn (What is the industrial use of the results obtained? What do the authors think is the greatest success of this research that can be introduced to the industry?)
Figure 1. and 2, 3
Pictures can not be on two separate pages, please move them to one side
I can not see the scale in the pictures, please add
Table 4 is very unreadable, please correct it - possibly add as supplementary file
Line 219 - "DCW-Y, L-Y, and Car-Y at different C/N ratios and nitrogen sources were shown in Table 3. The yield of DCW and carotenoid has reached a maximum at the third day and decreased significantly at the six-day, while the opposite was observed with lipid yield, this result also confirmed the reverse relationship between lipid and carotenoid production. " =Very poor conclusion, maybe more authors will describe.
Lack is of discussion and characteristics of other carotenoid-producing strains. Please check the latest article and add to introduction.
(2016) Rhodotorula glutinis-potential source of lipids, carotenoids, and enzymes for use in industries. Applied microbiology and biotechnology, 100 (14), 6103-6117.
"Impact of Aluminum sulfate on lipogenesis and carotenoid production" - There are not specific applications in this section. The authors should focus on discussing results with scientific literature and not just describing what they received!
Skillful presentation of the problem and description of the results together with a concrete discussion of the results (the latest literature) will interest the readers with this topic
After all, these studies require comparison. Please discuss the results and compare with the article:
(2019). Simultaneous Production of Lipids and Carotenoids by Red Yeast Rhodotorula from Waste Glycerol Fraction and Potato Wastewater. Applied Biochemistry and Biotechnology, 1-19. doi: 10.1007 / s12010-019-03023-z
A good discussion of the results with the latest literature and drawing conclusions, not speculation, should increase the interest in research in the scientific opinion in the world.
Author Response
Comment 1 The authors have not improved their admission again. There is not reference to the latest research.
(2016) Rhodotorula glutinis-potential source of lipids, carotenoids, and enzymes for use in industries. Applied microbiology and biotechnology, 100 (14), 6103-6117.
(2019). Simultaneous Production of Lipids and Carotenoids by Red Yeast Rhodotorula from Waste Glycerol Fraction and Potato Wastewater. Applied Biochemistry and Biotechnology, 1-19. doi: 10.1007 / s12010-019-03023-z
Answer: We appreciate the reviewer’s constructive suggestion. We read most of the latest research similar to this article and add it correctly to the introduction and discussion sections. For example：
1. Kot, A.M.; Błażejak, S.; Kurcz, A.; Gientka, I.; Kieliszek, M. Rhodotorula glutinis—potential source of lipids, carotenoids, and enzymes for use in industries. Appl. Microbiol. Biotechnol. 2016, 100, 6103–6117. https://doi.org/10.1007/s00253-016-7611-8
2. Zhu, Z.; Ding, Y.; Gong, Z.; Yang, L.; Zhang, S.; Zhang, C.; Lin, X.; Shen, H.; Zou, H.; Xie, Z.; Yang, F.; Zhao, X.; Liu, P.; Zhao, ZK. Dynamics of the lipid droplet proteome of the oleaginous yeast Rhodosporidium toruloides. Eukaryot Cell. 2015,14,252–264. https://doi.org/10.1128/EC. 00141-14
3. Sawangkeaw, R.; Ngamprasertsith, S. A review of lipid-based bio- masses as feedstocks for biofuels production. Renew Sust Energ Rev. 2013, 25(5):97–108. https://doi.org/10.1016/j.rser.2013.04.007
4. Kot, A.M.; Błażejak, S.; Kieliszek, M.; Gientka, I.; Bryś, J. Simultaneous Production of Lipids and Carotenoids by the Red Yeast Rhodotorula from Waste Glycerol Fraction and Potato Wastewater. Appl. Biochem. Biotechnol. 2019, 1-19. https://doi.org/10.1007/s12010-019-03023-z.
5. Jiru, T.M.; Groenewald, M.; Pohl, C.; Steyn, L.; Kiggundu, N.; Abate, D. Optimization of cultivation conditions for biotechnological production of lipid by Rhodotorula kratochvilovae(syn, Rhodosporidium kratochvilovae) SY89 for biodiesel preparation. 3 Biotech. 2017, 7:145. https://doi.org/10.1007/s13205-017-0769-7
Comment 2 Introduction, methodology and discussion of results should be a kind of correct history that smoothly goes to the conclusion. In the vale, it is worth describing the possibilities of producing lipids by the Rhodotorula strain. This information is missing. There are very few of them. Skillful presentation of the problem and description of the results together with a concrete discussion of the results (the latest literature) will interest the readers with this topic Lack is of discussion and characteristics of other carotenoid-producing strains. Please check the latest article and add to introduction. The authors can not find a link, draw conclusions because they do not read the latest scientific reports.
In the article, not only should new sentences be added in red but also conclusions should be drawn (What is the industrial use of the results obtained? What do the authors think is the greatest success of this research that can be introduced to the industry?) A good discussion of the results with the latest literature and drawing conclusions, not speculation, should increase the interest in research in the scientific opinion in the world.
Answer: We appreciate the reviewer’s constructive suggestion. We revised the manuscript.
For the introduction sections, the hypothesis of this article is outlined in the following: our studied strain Rhodotorula glutinis can produce highly valuable pigment –Torulene- under high C/N. Unfortunately, this condition is more favourable for lipid production, and from our work, we found that there is a reverse relationship between lipid and carotenoid production, which is similar to the previous studies, also. Accordingly, the optimization is detected as the aim of this research to state the best condition for enhancing torulene production over lipid production. To achieve this goal, we decide firstly to choose the best nitrogen source, the best C/S ratio and also C/N. Then study the effect of aluminium sulfate as a stress factor on the production of both lipid and carotenoid as an attempt to enhance carotenoid production as reported by the previous studies and at the same time study its effect on lipid accumulation.
In the introduction section, we wrote a brief description about all the important sides of our research which the reader will be interested about for example the importance of carotenoid, the carotenoid producing microorganisms, focusing on our studied yeast strain and its ability to produce lipid and carotenoid. Then give a brief description of torulene and why it is important to optimize its production — followed by describing the relationship between lipid and carotenoid production, finally talking briefly about the toxicity of aluminium ion on living organisms and its stimulatory role on carotenoid. The writing style and order of paragraph was carefully considered so the hypothesis and the aim of the research can be fully described and clarified to the reader.
When the introduction was written, most of the latest literature which correlated to our work was had the priority to mentioned first. For example, we removed reference 3, 4, and 5 and replaced them with more recent literature. Due to the shortage of knowledge regarding the effect of aluminium sulfate on the lipid pathway for oleaginous microorganism, we introduced some studies carried on plants and animals.
Worthy, a more recent literature were added to detect the possibilities of Rhodotorula glutinis to produce lipid in the introduction, result, and discussion.
For result and discussion sections, to facilitate the comparison of this study with the previous works, we decided to merge both discussion and result as one section. The writing style was carefully considered. The latest literature was introduced and compared with our work.
For conclusion, the conclusion section was rewritten to outline the most critical finding of this study and clarify the importance of this article to the industrial scale,
Comment 3 Figure 1. and 2, 3. Pictures can not be on two separate pages, please move them to one side. I can not see the scale in the pictures, please add.
Answer: Thanks for reviewer’s comment. All the figures and tables put properly on one page. Moreover, the not appeared scale bar was added to the figures; now, all scale bars can be easily distinguished.
Comment 4 Table 4 is very unreadable, please correct it - possibly add as a supplementary file
Answer: Thanks for reviewer’s comment. We added to table 4 as a supplementary data 1.
Comment 5: Line 219 - "DCW-Y, L-Y, and Car-Y at different C/N ratios and nitrogen sources were shown in Table 3. The yield of DCW and carotenoid has reached a maximum at the third day and decreased significantly at the six-day, while the opposite was observed with lipid yield, this result also confirmed the reverse relationship between lipid and carotenoid production. " =Very poor conclusion, maybe more authors will describe.
Answer: Thanks for reviewer’s comment. This sentence was rewritten to be " Kinetically, the reversed relationship between carotenoid and lipid production was observed when the yield and synthesis rate of both products were compared. As the highest carotenoid yield and synthesis rate was observed the third day while the highest lipid yield and synthesis rate were observed on the sixth date. The reverse relationship between lipid and carotenoid production by red oleaginous yeasts was also reported by [10,23]"
Comment 6 "Impact of Aluminum sulfate on lipogenesis and carotenoid production" - There are not specific applications in this section. The authors should focus on discussing results with scientific literature and not just describing what they received!
Answer: We appreciate the reviewer’s constructive suggestion. The difficulties which we faced while discussing the results of these sections is the shortage of literature which illustrate the effect of aluminium ion on both lipid and carotenoid production by red yeasts. We discussed our result depends on literature which studying the effect of aluminium on phospholipid and lipid synthesis pathway in plants and animals, and other stress condition like irradiation and heavy metal generally on carotenoid productions by red yeasts. The latest literature was added and compared with our work.
Comment 7 After all, these studies require comparison. Please discuss the results and compare it with the article.
Answer: We appreciate the reviewer’s constructive suggestion. In the article, we compared all the critical finding with their similar previous work and also introduced table 6 to compare the yield of carotenoid and torulene as well, between our work and the previously reported studies. The studies which we used for comparison used glucose as a carbon source plus growing the yeast strain in shaken flask fermentation. In this table, we focus mainly on torulene and carotenoid as the main aim of this study is maximizing torulene production.
Round 3
Reviewer 3 Report
The article has been corrected. I recommend publication for acceptance.
