Growth Performance and Biochemical Composition of Waste-Isolated Microalgae Consortia Grown on Nano-Filtered Pig Slurry and Cheese Whey under Mixotrophic Conditions

Round 1

Reviewer 1 Report

This manuscript investigated growth performance and biochemical composition of waste-isolated microalgae consortia grown on nano-filtered pig slurry and cheese whey under mixotrophic conditions. The obtained results would provide data support to large-scale cultivation of microalgae consortia using wastewater. In my view, this research topic falls in the scope of Fermentation, and the research results are interesting. I think this paper will be of particular interest to those working in this field. 

Specific comments:

1. The expression of data should follow the scientific rules, such as the consist of the digitals of the data.

2. In Materials and methods part, more details should be given. Thus, readers can easily to repeat the results in the manuscript. For example, what was the inoculation ratio ? 

3. Why was the cheese whey stored at -20 ⁰C after collection?

4. In Table 3, some data are reported as means ± standard deviation. The others are reported as means. Please revise the table. 

5.In Table 4, one-way ANOVA of proteins and lipids was carried out. However, there were no one-way ANOVA of carbohudrates. Please revise the table. 

Author Response

REVIEWER 1:

Open Review

( ) I would not like to sign my review report
(x) I would like to sign my review report

English language and style

( ) Extensive editing of English language and style required
( ) Moderate English changes required
(x) English language and style are fine/minor spell check required
( ) I don't feel qualified to judge about the English language and style

Comments and Suggestions for Authors

This manuscript investigated growth performance and biochemical composition of waste-isolated microalgae consortia grown on nano-filtered pig slurry and cheese whey under mixotrophic conditions. The obtained results would provide data support to large-scale cultivation of microalgae consortia using wastewater. In my view, this research topic falls in the scope of Fermentation, and the research results are interesting. I think this paper will be of particular interest to those working in this field. 

Specific comments:

1. The expression of data should follow the scientific rules, such as the consist of the digitals of the data.

WE: The reviewer is right. Please find corrected table 1, table 4, figure 1c, and data expression in text.

2. In Materials and methods part, more details should be given. Thus, readers can easily to repeat the results in the manuscript. For example, what was the inoculation ratio?

WE: The reviewer is right. Please find the inoculation ratio and more details in section 2.4

3. Why was the cheese whey stored at -20°C after collection?

WE: The cheese whey was stored at -20°C because it allows preserving and miming the availability of fresh cheese whey for a long time since there were many batch trials and limited batch reactors in our lab. It provided us the opportunity to collect the cheese way once and to use the same stock (stored as several aliquots that could be defreezed at the time of use) for all the trials, preserving the chemical and nutritional properties. Storing cheese whey at 4 °C does not allow preserving it for long time unless it was sterilized at high temperature modifying characteristics. Therefore, all characteristics and daily availability of fresh cheese whey can be guaranteed at -20°C.

In Table 3, some data are reported as means ± standard deviation. The others are reported as means. Please revise the table.

WE:  The reviewer is right. The modified table 3 and the annotate can be found after checking the data. Some data (e.g., TN_final, N_biomass, N uptaken by biomass, P_final, P uptaken by biomass, C_final, TOC removal) of AC_11 does not have standard deviation because the replicated culture failed.

4. In Table 4, one-way ANOVA of proteins and lipids was carried out. However, there were no one-way ANOVA of carbohydrates. Please revise the table. 

WE: The reviewer is right. Please find modified table 4.

Reviewer 2 Report

The work is of good quantity and quality, some minor revisions needed before accepting the manuscript.

1.  The figure 1  and figure 2 - check the spelling of the amino acids, they need to corrected in the figure itself.

2. Provide some sort of explanation as to why the mixotrophic cultivation of consortium 3 and 10 crashed. because they are cyanobacteria dominant? 

Author Response

REVIEWER 2:

Open Review

( ) I would not like to sign my review report
(x) I would like to sign my review report

English language and style

( ) Extensive editing of English language and style required
( ) Moderate English changes required
(x) English language and style are fine/minor spell check required
( ) I don't feel qualified to judge about the English language and style

Comments and Suggestions for Authors

The work is of good quantity and quality, some minor revisions needed before accepting the manuscript.

1. The figure 1 and figure 2 - check the spelling of the amino acids, they need to corrected in the figure itself.

WE: The reviewer is right. Please find the revised figure 1a and figure 2a.

2. Provide some sort of explanation as to why the mixotrophic cultivation of consortium 3 and 10 crashed. because they are cyanobacteria dominant? 

WE: The reviewer is right. Some explanation can be found in section 3.2.

Author Response File: Author Response.docx

Reviewer 3 Report

General comments:

This manuscript delineates the growth ability of twelve algae-microbial consortia (ACs) originally selected from organic wastes when nano-filtered pig slurry wastewater (NFP) and cheese whey (CW) were used as growth substrates in mixotrophic mode in comparison with photoautotrophic mode. On the whole, the content of the article is comprehensive, but there is a lack of summary conclusions, such as which AC has the best nutrient removal? And using algae-microbial consortia to treat mixed wastewater, which not an interesting or novel topic. Moreover, the experiment design and manuscript organization need improvement. Other problems to be solved are as follows:

1. Confused, what is the initial inoculum amount for AC? How did the authors ensure that the AC inoculum was consistent and uniform, and how was it measured? Please explain in details.

2. Literature survey is not sufficient to present the most updated status about wastewater treatment with microalgae. The author should analyze the articles related to this issue in recent two years and strengthen the importance their research.

3. The language is not refined enough and the long sentence is too difficult to understand, such as line 71-75: “Rather than attempting to combat the contamination of monocultures by unwanted and detrimental species, cultivating microalgae consortia of microalgae and bacteria is a good solution, as they are efficient in detoxifying organic and inorganic pollutants, and removing nutrients from wastewater, compared to the individual microorganisms” and line 79-83: “The growth performance, nutrient recovery ability, and biochemical composition accompanying fatty acids and amino acids profile in the mixotrophic mode were evaluated and compared with autotrophic conditions when the cultures were supplied with only an NFP medium, aiming to understand whether changing cultivation mode affects the growth performance and biomass quality of algae consortia”. The English language should be improved to ensure that an international audience can clearly understand your text. The current phrasing makes comprehension difficult.

Keywords:

4. the keyword “Nutrient recovery” is inaccuracy, it is recommended to be modified.

Introduction:

5. Line 61: “ammonium N” should be “ammonium (NH3-N)”.

6. Line 65: “by-products” should be “bio‑product”.

Materials and Methods:

7. Line 90-96:What is the role of S1, S2, L1, L2? Are these useful in subsequent experiments? If not, it is recommended to delete.

8. Line 97: “more than 80% is ammonium-N”, How did the author get it? What is the specific concentration?

9. Line 103: “Total nitrogen (TN), ammonia nitrogen (NH4+-N), pH and chemical oxygen demand (COD)…” should be “Total nitrogen (TN), ammonia nitrogen (NH4+-N), pH, and chemical oxygen demand (COD)…”.

10. 2.5. Microalgae growth determination: In addition to microalgae, there are other organisms in AC. Can the microalgae dry weight method represent the growth of AC? This method is inaccurate.

11. Line 165: “total N” should be “TN”.

12. Line 205: “the microalgae oils” means “biodiesel”?

13. Line 228, 240: “C” and “P” cannot be abbreviated. Please check the full text.

Figture:

14. The figtures should be standard.

Author Response

REVIEWER 3:

Open Review

( ) I would not like to sign my review report
(x) I would like to sign my review report

English language and style

( ) Extensive editing of English language and style required
(x) Moderate English changes required
( ) English language and style are fine/minor spell check required
( ) I don't feel qualified to judge about the English language and style

Comments and Suggestions for Authors

General comments:

This manuscript delineates the growth ability of twelve algae-microbial consortia (ACs) originally selected from organic wastes when nano-filtered pig slurry wastewater (NFP) and cheese whey (CW) were used as growth substrates in mixotrophic mode in comparison with photoautotrophic mode. On the whole, the content of the article is comprehensive, but there is a lack of summary conclusions, such as which AC has the best nutrient removal? And using algae-microbial consortia to treat mixed wastewater, which not an interesting or novel topic. Moreover, the experiment design and manuscript organization need improvement. Other problems to be solved are as follows:

WE: The reviewer is right. Please find revised conclusion.

1. Confused, what is the initial inoculum amount for AC? How did the authors ensure that the AC inoculum was consistent and uniform, and how was it measured? Please explain in details.

WE: Please find modified detailed information in section 2.4.

2. Literature survey is not sufficient to present the most updated status about wastewater treatment with microalgae. The author should analyze the articles related to this issue in recent two years and strengthen the importance their research.

WE: The reviewer is right. Please find modified text in introduction section.

3. The language is not refined enough and the long sentence is too difficult to understand, such as line 71-75: “Rather than attempting to combat the contamination of monocultures by unwanted and detrimental species, cultivating microalgae consortia of microalgae and bacteria is a good solution, as they are efficient in detoxifying organic and inorganic pollutants, and removing nutrients from wastewater, compared to the individual microorganisms” and line 79-83: “The growth performance, nutrient recovery ability, and biochemical composition accompanying fatty acids and amino acids profile in the mixotrophic mode were evaluated and compared with autotrophic conditions when the cultures were supplied with only an NFP medium, aiming to understand whether changing cultivation mode affects the growth performance and biomass quality of algae consortia”. The English language should be improved to ensure that an international audience can clearly understand your text. The current phrasing makes comprehension difficult.

WE：Please find the modified text in line 73-77 and line 81-85. Moreover, the text has been reviewed by mother tongue reviewer and correctio  

Keywords:

4. the keyword “Nutrient recovery” is inaccuracy, it is recommended to be modified.

WE：Please find modified keywords.

Introduction:

5. Line 61: “ammonium N” should be “ammonium (NH3-N)”.

WE：Please find modified text.

6. Line 65: “by-products” should be “bio‑product”.

WE：Thank you, but we meant “by-product” in this context.

Materials and Methods:

7. Line 90-96: What is the role of S1, S2, L1, L2? Are these useful in subsequent experiments? If not, it is recommended to delete.

WE：The reviewer is right. We deleted this part.

8. Line 97: “more than 80% is ammonium-N”, How did the author get it? What is the specific concentration?

WE：This data (that more than 80% TKN of output liquid fraction produced in the process (for getting NFP) is in ammonium-N form) was obtained from the pig slurry treatment plant. Please see modified part in section 2.1.

9. Line 103: “Total nitrogen (TN), ammonia nitrogen (NH4+-N), pH and chemical oxygen demand (COD)…” should be “Total nitrogen (TN), ammonia nitrogen (NH4+-N), pH, and chemical oxygen demand (COD)…”.

WE：Please find the modified text.

10. 5. Microalgae growth determination: In addition to microalgae, there are other organisms in AC. Can the microalgae dry weight method represent the growth of AC? This method is inaccurate.

WE: The reviewer is right. We meant for microalgae consortia biomass growth including the microorganisms. The microalgae growth determination has been modified to microalgae consortia growth determination.

11. Line 165: “total N” should be “TN”.

WE: Please find the modified text.

12. Line 205: “the microalgae oils” means “biodiesel”?

WE: “The microalgae oils” means “the microalgae lipids” in this context. Please find the modified text.

13. Line 228, 240: “C” and “P” cannot be abbreviated. Please check the full text.

WE: The reviewer is right. We specified C in new line 30. P was specified in line 53. The full text has been checked.

Figure:

14. The figures should be standard.

WE: Please find modified figures.

Round 2

Reviewer 1 Report

The questions in the original manuscript have been well revised. It is suitable for the possible publication.

Author Response

The questions in the original manuscript have been well revised. It is suitable for the possible publication.

We: Thank you

Reviewer 3 Report

Using algae-microbial consortia to treat mixed wastewater is not an interesting or novel topic. The scientific significance of this study is not clear.

And the authors did not respond to all comments. Other problems to be solved are as follows:

1.     The problem about the initial inoculum amount for AC has not been solved. I asked how to ensure the consistency and uniformity of AC inoculum and how to measure it? Please explain in detail.

2.     No improvement in the language. Please get a professional to correct the manuscript and attach proof.

3.     “more than 80% is ammonium-N”. This is a rigorous process. You need to measure it, not obtain the approximate value from the pig slurry treatment plant.

4.     Figures: not modified with high quality, just enlarged?

Author Response

Using algae-microbial consortia to treat mixed wastewater is not an interesting or novel topic. The scientific significance of this study is not clear.

WE: We agree that using algae-microbial consortia to treat mixed wastewater is not a new topic. But this research is the follow-up study from isolating indigenous algae-microbial consortia from sixteen organic wastes [1], to be then used for technological applications to treat organic wastes in different modes i.e. autotrophic mode [2], and mixotrophic mode (this paper), then further to be used in large scale cultivation.

The first novelty lies in the origins of twelve algae consortia (ACs) used in this study. They are indigenous, autochthonous algae-microbial consortia isolated from sixteen various organic wastes sampled in north Italy such as reported by previous work [1]. These ACs were then isolated and tested to treat organic wastes removing nutrients and producing useful biomass in different modes i.e. autotrophic mode [2], and mixotrophic mode (this paper) in batch scale. The results related to growth performance, nutrient recovery ability and biochemical compositions obtained from this study could provide essential information and guidance for large scale cultivation. In addition, this kind of selection could be a process upgrade for microalgae-based wastewater treatment plants since these rely on spontaneous algae bloom at present.

This part has been addressed and re-addressed in the introduction and in the conclusion.

[1].      Su, M.; Dell’Orto, M.; D’Imporzano, G.; Bani, A.; Dumbrell, A.J.; Adani, F. The Structure and Diversity of Microalgae-Microbial Consortia Isolated from Various Local Organic Wastes. Bioresour. Technol. 2022, 347, 126416, doi:10.1016/j.biortech.2021.126416.

[2].      Su, M.; Dell’orto, M.; Scaglia, B.; D’imporzano, G.; Bani, A.; Adani, F. Growth Performance, Biochemical Composition and Nutrient Recovery Ability of Twelve Microalgae Consortia Isolated from Various Local Organic Wastes Grown on Nano-Filtered Pig Slurry. Molecules 2022, 27, 1–19, doi:10.3390/molecules27020422.

And the authors did not respond to all comments. Other problems to be solved are as follows:

1. The problem about the initial inoculum mount for AC has not been solved. I asked how to ensure the consistency and uniformity of AC inoculum and how to measure it? Please explain in detail.

WE: the reviewer is right, we tried to eliminate the confusion, better explaining the procedure to obtain a suitable inoculum to start batch cultures. For what concerns the quantitative aspect, we obtained algae-microbial consortia inocula with a dry biomass concentration between 2 and 3 g L^-1, in order to obtain, after dilution 1:10 in the batch (Section 2.4), a dry biomass initial concentration between 0.2 g L^-1 and 0.3 g L^-1. The AC dry biomass concentration of the inoculum was determined according to the method reported in Section 2.5. For what concerns the qualitative aspect, i.e. consistency and stability of the inoculum, we performed all the batch trials in a small range of time, just after having performed the NGS analysis on the AC cultures used as inocula [1]. For this reason, we assumed that, leaving unchanged the growth conditions, the eukaryotic and prokaryotic composition of the inocula would not change so much.

We are aware that this is a weak point, and that the stability of the inoculum should be assessed, for this reason we have planned to perform again NGS analysis on those ACs inocula that will be actually used for pilot-scale trials.

This part had been modified in section 2.4.

2. No improvement in the language. Please get a professional to correct the manuscript and attach proof.

WE: paper has been corrected both at first and second round by mother tongue scientific reviewer. We do another time and in attach also certificate. 

3. “more than 80% is ammonium-N”. This is a rigorous process. You need to measure it, not obtain the approximate value from the pig slurry treatment plant.

WE: Please find modified text in section .2.1.

4. Figures: not modified with high quality, just enlarged?

WE: Thank you. However, the figures were directly extracted from excel, so the quality of figures is fixed.

Accept after minor revision

Dear Authors, Although the paper has been considered interesting, there are many comments that must be addressed including the revision of the English language. Decimals, SD must be included in ALL tables and graphs, wide explanations, highlight the novelty and the results, ETC. Please, the whole document must be carefully revised.

WE: Please find the supplementary material Table 2S: the standard deviation data for figure 1a (amino acids). However, for figure 1b and 1c (fatty acids), unfortunately we cannot obtain the standard deviation because we do not have enough materials to obtain the replicates.

Author Response File: Author Response.docx