Agronomic and Environmental Performance of Lemnaminor Cultivated on Agricultural Wastewater Streams—A Practical Approach

Round 1

Reviewer 1 Report

The topic of work interesting and up-to-date. Comparing different substrates (composition of the influent cubicontainer), the Authors demonstrated the legitimacy of their use in the production of biomass from duckweed. It is important to monitor the quality of the substrates used and the climatic conditions with the growth of duckweed. Despite the practical aspect contained in the title (A Practical Approach), the authors did not referred to duchweed harvesting and processing methods as an important source of protein for livestock.

The Authors showed that the biological value of the duckweed protein and its amino acid composition is not ideal for different species of livestock, needs to be supplemented with other feed additives, but can provide a source of valuable protein as a replacement for soybean meal GMOs.

L 232: "For pigs and sows..."this is the same species, please change to "for growing pigs and sows..."

L 770: should be: D. Pieniążek, M. Rakowska, W. Szkiłładziowa, Z. Grabarek

Author Response

Thank you for your revision. Please see the attachment for the point-by-point respons. 

Author Response File: Author Response.pdf

Reviewer 2 Report

GENERAL COMMENTS

 Comments:  The work is interesting. It proposes a pilot-scale system to grow Lemna minor under outdoor conditions to treat different wastewaters to produce protein-enriched material

 Comments:  The paper provides data from a 175-day trial in outdoor conditions, which is of high relevance for the scale-up of the technology.

Comments:  The Authors compare the results of the two wastewaters with a synthetic culture medium. However, the form of the nutrients present should be better explained.

Comments:  The authors intend to produce a value-added product, rich in protein as an alternative to others on the market. However, its use in human food complies with very high food safety criteria, which should be reconsidered.

Abstract: is Ok

INTRODUCTION

Comments:  - the state of the art is adequate and contextualizes the problem

METODOLOGY:

Comments:  The methods are well described.

Comments: The Authors compare the results of the two wastewaters with a synthetic culture medium. However, although the total nitrogen content is similar in the different culture media, the forms of nitrogen are different, with different speeds of assimilation by the plants, and may give rise to doubts of interpretation. Similarly, the different forms of phosphorus are not evident.

RESULTS

Comments:  In Table 1 the abbreviation of the growing medium should be shown, because in the legend it appears “diluted pig manure wastewater” and in the table, the same culture medium is “biological effluent”. Also in line 299 the authors mentioned swine manure wastewater. Please harmonize...

Comments:  Line 304 explain why NH4-N is within maximal ranges. Is it a typo? According to Table 1 this value seems to be lower than the other means. Confused.

Comments:  The authors does not provided information on P forms, orthophosphate, inorganic P and organic P. This information should be added to support discussion. Without this information authors cannot justify lines 327-329.

Comments:  The authors should justify the lack of data in the BE assay (Figure 1) for higher temperature.

Comments:  There is a Figure in Appendix whose abbreviation should be changed: PE should be PP. The Figure is also numbered as Figure 2 and causes confusion with the other Figure 2.

DISCUSSION

Comments:  Regarding die-off of duckweed on BE, authors mention the existence of a toxic compound whose effect lasted for 15 days or 2 retention times (line 455). However, in line 127-128 the retention times are about 25 days and 9 hours. It needs to be reviewed.

Comments:  The authors suggest that this toxicity may be related to ammoniacal nitrogen and pH. It would be interesting for the authors to introduce the graphs of the PP assay in Figure 2, as the justification will be enriched by a comparison of an assay that did not suffer any negative effect on duckweed growth, but whose average ammoniacal nitrogen concentration was similar (Table 1).

Comments:  Line 490-493 After treatment, a storage lagoon does not have a buffer effect. The authors are considering recirculation? Other suggestion? The authors should further specify this statement.

Author Response

Thank you for your revision. Please see the attachment for the point-by-point respons. 

Author Response File: Author Response.pdf

Reviewer 3 Report

The authors address a very important question: large-scale cultivation of duckweed under outdoor conditions using wastewater. This is actually the bottleneck in practical applications of duckweeds for feed, food and as energy plant as well as for wastewater cleaning. The experiments were well planned and carried out as well as evaluated. I have only remarks in details and suggest therefore accepting after minor revision.

1. The first sentence of the Abstract does not have the character of an abstract. I suggest to start with the second sentence and integrate the intention of the first sentence in the second one.
2. Lines 19-20: Abbreviations PP and BE: Are these effluents not both biological effluents? If the authors agree, then perhaps PM for pig manure would be better?
3. Line 24: Abstract: “regardless of the used media”. Can it be concluded from the data that the nutrient medium/wastewater hat rather influence on the protein content but rarely on the AA composition? If so, this should be clearly written.
4. Page 2, line 48: The term Lemnoideae became a difficult term. It could be first use as a term for the subfamily of duckweed including the genera Spirodela, Landoltia and Lemna. Use the term with this meaning would make sense as the authors investigated just one species from this “subfamily”. When it is used meaning the whole of duckweed as a subfamily of Araceae – this would be justified as APG uses it in this sense but would be in contrast to the overwhelming majority of duckweed publications. To make this controversy clear in a publication about Lemna minor would be a little bit complicated. I suggest therefore to cancel the Latin term and just using “duckweed”.
5. Page 2, line 50: “Are one of the most rapidly growing” – do the authors know a plant species that growth faster than duckweed?
6. Line 60: “dairy wastewater”: I suggest consulting the paper of Walsh et al. J. Phytorem. 22: 694 (2020) as these authors make troubleshooting in experiments with dairy waste.
7. Page 2, line 66: “Moreover, several studies determined the protein content of duckweed, but few 66 have analysed its amino acid (AA) composition…” I suggest consulting the paper of Appenroth et al. Frontiers Chemistry 6: 483 (2018) and references therein, as not only a larger number of species were investigated but also all essential amino acids (cf. chapter 2.4.2 of the present manuscript).
8. Page 3: “mineral nutrient composition for duckweed cultivation, which is described in the duckweed ISCDRA forum volume 3 [11,24]”. The reference should be given correctly. Moreover, in the mentioned article of the “Duckweed Forum” several useful media were compiled. Which of these media was used in the present project? Table A2: Which medium was used for the present investigations?
9. Chapter 2.4.2. This is a flaw, it would have been better to analyse all amino acids.
10. Page 8, line 304-305: Do you mean within minimal range? Is there a minimal requirement for Na+?
11. Page 8, lines 306-308: I am sure that a high level of EC cannot compensate for a lack of any specific nutrient element, also not for NH4-N.
12. Page 11, line 385: Is the anyone who doubts the usefulness of the Kjeldahl method for duckweed?
13. Page 11, line 399-402: If this is correct then make clearer that there is hardly any environmental effect on the AA composition (see also abstract).
14. Page 12, line 440-424: I suggest to be more careful with this comparison. Is there any proof that humic acids have a stimulatory effect in the aquatic system?
15. Page 14, line 474-481: If I understood correctly then I suggest to mention hat the very reason is not really known.

    The authors address a very important question: large-scale cultivation of duckweed under outdoor conditions using wastewater. This is actually the bottleneck in practical applications of duckweeds for feed, food and as energy plant as well as for wastewater cleaning. The experiments were well planned and carried out as well as evaluated. I have only remarks in details and suggest therefore accepting after minor revision.

    1. The first sentence of the Abstract does not have the character of an abstract. I suggest to start with the second sentence and integrate the intention of the first sentence in the second one.
    2. Lines 19-20: Abbreviations PP and BE: Are these effluents not both biological effluents? If the authors agree, then perhaps PM for pig manure would be better?
    3. Line 24: Abstract: “regardless of the used media”. Can it be concluded from the data that the nutrient medium/wastewater hat rather influence on the protein content but rarely on the AA composition? If so, this should be clearly written.
    4. Page 2, line 48: The term Lemnoideae became a difficult term. It could be first use as a term for the subfamily of duckweed including the genera Spirodela, Landoltia and Lemna. Use the term with this meaning would make sense as the authors investigated just one species from this “subfamily”. When it is used meaning the whole of duckweed as a subfamily of Araceae – this would be justified as APG uses it in this sense but would be in contrast to the overwhelming majority of duckweed publications. To make this controversy clear in a publication about Lemna minor would be a little bit complicated. I suggest therefore to cancel the Latin term and just using “duckweed”.
    5. Page 2, line 50: “Are one of the most rapidly growing” – do the authors know a plant species that growth faster than duckweed?
    6. Line 60: “dairy wastewater”: I suggest consulting the paper of Walsh et al. J. Phytorem. 22: 694 (2020) as these authors make troubleshooting in experiments with dairy waste.
    7. Page 2, line 66: “Moreover, several studies determined the protein content of duckweed, but few 66 have analysed its amino acid (AA) composition…” I suggest consulting the paper of Appenroth et al. Frontiers Chemistry 6: 483 (2018) and references therein, as not only a larger number of species were investigated but also all essential amino acids (cf. chapter 2.4.2 of the present manuscript).
    8. Page 3: “mineral nutrient composition for duckweed cultivation, which is described in the duckweed ISCDRA forum volume 3 [11,24]”. The reference should be given correctly. Moreover, in the mentioned article of the “Duckweed Forum” several useful media were compiled. Which of these media was used in the present project? Table A2: Which medium was used for the present investigations?
    9. Chapter 2.4.2. This is a flaw, it would have been better to analyse all amino acids.
    10. Page 8, line 304-305: Do you mean within minimal range? Is there a minimal requirement for Na+?
    11. Page 8, lines 306-308: I am sure that a high level of EC cannot compensate for a lack of any specific nutrient element, also not for NH4-N.
    12. Page 11, line 385: Is the anyone who doubts the usefulness of the Kjeldahl method for duckweed?
    13. Page 11, line 399-402: If this is correct then make clearer that there is hardly any environmental effect on the AA composition (see also abstract).
    14. Page 12, line 440-424: I suggest to be more careful with this comparison. Is there any proof that humic acids have a stimulatory effect in the aquatic system?
    15. Page 14, line 474-481: If I understood correctly then I suggest to mention hat the very reason is not really known.

        The authors address a very important question: large-scale cultivation of duckweed under outdoor conditions using wastewater. This is actually the bottleneck in practical applications of duckweeds for feed, food and as energy plant as well as for wastewater cleaning. The experiments were well planned and carried out as well as evaluated. I have only remarks in details and suggest therefore accepting after minor revision.

        1. The first sentence of the Abstract does not have the character of an abstract. I suggest to start with the second sentence and integrate the intention of the first sentence in the second one.
        2. Lines 19-20: Abbreviations PP and BE: Are these effluents not both biological effluents? If the authors agree, then perhaps PM for pig manure would be better?
        3. Line 24: Abstract: “regardless of the used media”. Can it be concluded from the data that the nutrient medium/wastewater hat rather influence on the protein content but rarely on the AA composition? If so, this should be clearly written.
        4. Page 2, line 48: The term Lemnoideae became a difficult term. It could be first use as a term for the subfamily of duckweed including the genera Spirodela, Landoltia and Lemna. Use the term with this meaning would make sense as the authors investigated just one species from this “subfamily”. When it is used meaning the whole of duckweed as a subfamily of Araceae – this would be justified as APG uses it in this sense but would be in contrast to the overwhelming majority of duckweed publications. To make this controversy clear in a publication about Lemna minor would be a little bit complicated. I suggest therefore to cancel the Latin term and just using “duckweed”.
        5. Page 2, line 50: “Are one of the most rapidly growing” – do the authors know a plant species that growth faster than duckweed?
        6. Line 60: “dairy wastewater”: I suggest consulting the paper of Walsh et al. J. Phytorem. 22: 694 (2020) as these authors make troubleshooting in experiments with dairy waste.
        7. Page 2, line 66: “Moreover, several studies determined the protein content of duckweed, but few 66 have analysed its amino acid (AA) composition…” I suggest consulting the paper of Appenroth et al. Frontiers Chemistry 6: 483 (2018) and references therein, as not only a larger number of species were investigated but also all essential amino acids (cf. chapter 2.4.2 of the present manuscript).
        8. Page 3: “mineral nutrient composition for duckweed cultivation, which is described in the duckweed ISCDRA forum volume 3 [11,24]”. The reference should be given correctly. Moreover, in the mentioned article of the “Duckweed Forum” several useful media were compiled. Which of these media was used in the present project? Table A2: Which medium was used for the present investigations?
        9. Chapter 2.4.2. This is a flaw, it would have been better to analyse all amino acids.
        10. Page 8, line 304-305: Do you mean within minimal range? Is there a minimal requirement for Na+?
        11. Page 8, lines 306-308: I am sure that a high level of EC cannot compensate for a lack of any specific nutrient element, also not for NH4-N.
        12. Page 11, line 385: Is the anyone who doubts the usefulness of the Kjeldahl method for duckweed?
        13. Page 11, line 399-402: If this is correct then make clearer that there is hardly any environmental effect on the AA composition (see also abstract).
        14. Page 12, line 440-424: I suggest to be more careful with this comparison. Is there any proof that humic acids have a stimulatory effect in the aquatic system?
        15. Page 14, line 474-481: If I understood correctly then I suggest to mention hat the very reason is not really known.v

Author Response

Thank you for your thorough review. Please see the attachment for a point-by-point response. 

Author Response File: Author Response.pdf

Reviewer 4 Report

Dear Authors,

thank you for sending me a very interesting manuscript. In fact, I have no major comments on the text.

Yours faithfully,

Author Response

Dear reviewer, 

Thank you very much for the positive feedback. This is much appreciated. 

Sincerely, 

Reindert Devlamynck

Round 2

Reviewer 2 Report

The changes have been taken into account and the paper has been greatly improved.
Congratulations!