Reducing the Phytoplankton Biomass to Promote the Growth of Submerged Macrophytes by Introducing Artificial Aquatic Plants in Shallow Eutrophic Waters

Round 1

Reviewer 1 Report

The manuscript entitled “Reducing phytoplankton biomass to promote the growth of submerged macrophytes by introducing artificial aquatic plants in a shallow eutrophic lake” by Wu et al. presents a unique mesocosm experiment investigating the impacts of artificial aquatic plants on the reestablishment of macrophytes in a eutrophic shallow lake.  The manuscript is well written and the experiment is well designed and documented.  I do think the data set and experiment is publishable, but the manuscript in its current form has some extreme flaws that the authors must address.  I have listed these below as major comments followed by minor comments on the manuscript.

Major Comments:

1)    The mesocosms are far too small to extrapolate the results to entire lake systems.  The dimensions given by the authors show that the enclosures are only 36m2 which is 0.000012% of the lake surface area.  In addition, the mesocosm were in 2 m of water which is less than have of the average depth of the lake.  In no way, can these results be applied to whole lake systems.  What the authors have shown is that artificial plants can aid in macrophyte reestablishment in littoral areas.  After that, they cannot apply this technology.  They have no evidence or data to support the application to an entire shallow lake.  As a result, I would suggest the authors go back through the and remove text suggesting this overstep of application.  I would suggest a section at the end of the manuscript discussing limitations and applications of this idea.  What about the use of these artificial plants in aquaculture establishments?  It appears that application could be supported with their data since aquaculture establishments use small enclosures.   

2)    The second major flaw of the paper is the complete neglect of sediment resuspension.  It is known that resuspended sediments are a primary source of internal loading of nutrients and a major feedback of macrophytes are the anchoring of sediments.  The enclosures used in this study would stop resuspension since they were open on the top and bottom and prevented wind impacts.  Looking at the data, NTU, COD, TP and chl-a all decreased in the controls which could be attributed somewhat to the prevention of sediment resuspension by the enclosures.  In addition, the authors do not measure sediments in their analyses.  While they attribute the artificial plants as taking up an extrodanary amount of P, they neglected to show if in the controls, the P sedimented out from dead phytoplankton. In addition, there mechanism does not include sediments as a primary factor in these systems.  This is incorrect and anyone who is studying shallow lakes must account for sediment processes.  Please read all of the work Martin Scheffer has done on this subject including his book on shallow lake ecology.  Since the authors did not measure sediments in this study, they must discuss that as a possible mechanism throughout the text to apply these findings to any shallow system.

3)    If this technology is going to be applied to other systems, a breakdown of cost needs to be included.  There is no other way to need this systems except in management situations.  As a result, cost must be discussed and hopefully described somewhere in the text.

Minor comments:

Lines 1-4:  The title must be changed because in no way do the authors proved that this mechanism would work in an entire lake system.  I would change “in a shallow eutrophic lake” to “shallow waters”.

Lines 34-35: the role of macrohpytes to anchor sediments needs to be included from the onset.

Line 68: “previous studies” are mentioned but neither cited or explained.  You can’t just justify something as “previous studies” you need to show the reader how you can say that statement.

Line 91: at 4.4 m mean depth, is this a shallow lake?  Seems deeper than a shallow lake.  Again, I do not think the results from this study can be extrapolated to the rest of the lake.  You do not have data to show that.

A table needs to be included giving more detail of the lake conditions.  Do not send the reader to a paper.  The entire paper is about the changes in lake conditions so please give the reader starting conditions.  N, P, secchi, chl-a, z-max, and anything else needs to be included.  For a paper based on fixing the lake (even in the title), little is provided about the lake itself.

Line 98:  Figure 1 is terrible.  I cannot make out what is the lake and what is the watershed.  Where were the enclosures?  Are the words, study site by the mesocosm site or is that label for the entire map?  The right and bottom inset is not helpful as tall.  Finally, the Figure description is too basic.  Please help the reader see what is needed here.

Lines: 158-172: the measurement of bacteria is a surprise to the reader.  It needs to be stated what is the question and use of this data.

Lines191-213: Again, a table is needed here.  There is no way a reader can truly get the information among the statements and “respectfully’s”. 

Line 213: stating that this can be applied to all eutrophic waters is an overstatement.  Maybe, shallow littoral areas or small eutrophic ponds such as agricultural impoundments or aquaculture ponds.

Lines 261-263: The figure description needs to be expanded.  What is CW0, CW1 or TB1, TB2. 

Lines 314-320:  What if diversity increased in the sediments of the control?  This is not measured and is a viable place for microbial processes.

Line 343: change didn’t to did not

Line 365: Sediments need to be included in the conceptual figure.

Line 372: again, eutrophicated lakes is an over statement for the data provided.  Change this to a more similar application (shallow littoral areas or small eutrophic ponds such as agricultural impoundments or aquaculture ponds)

Author Response

Major Comments:

1) The mesocosms are far too small to extrapolate the results to entire lake systems.  The dimensions given by the authors show that the enclosures are only 36m2 which is 0.000012% of the lake surface area.  In addition, the mesocosm were in 2 m of water which is less than have of the average depth of the lake.  In no way, can these results be applied to whole lake systems.  What the authors have shown is that artificial plants can aid in macrophyte reestablishment in littoral areas.  After that, they cannot apply this technology.  They have no evidence or data to support the application to an entire shallow lake.  As a result, I would suggest the authors go back through the and remove text suggesting this overstep of application.  I would suggest a section at the end of the manuscript discussing limitations and applications of this idea.  What about the use of these artificial plants in aquaculture establishments?  It appears that application could be supported with their data since aquaculture establishments use small enclosures.   

Thank you for your good advice.

First, taking the current design of the experiment, we would express carefully the result to entire Dianchi lake systems, so we would take your suggestion for changing“in a shallow eutrophic lake” to“shallow eutrophic waters”. While, in the successive design, we hope to investigate a large-scale experiment to explore the feasibility of periphyton biofilms attached on AAPs to reduce cyanobacteria blooms in a suitable lake. And at the end of the manuscript, we would give discussion about limitations and applications of this idea. Such as, we envision to encounter some problems during the transfer of the small-scale experiments reported here to a whole lake situation. Technical problems to expose and safely remove of thousands m² AAPs need to be solved. Human interference also needs to be taken into consideration. Importantly, the large-scale introduction of AAPs and hence the growth of periphyton may influence the ecosystem in a profound and perhaps unpredictable way.

Additionally, the article stated that artificial plants can aid in macrophyte reestablishment in enclosures of freshwater lake rather than littoral areas and provide potential for the application of AAPs was an environmentally-friendly and effective solution for promoting the restoration of submerged macrophytes in shallow eutrophic lakes.  

Such as, we envision to encounter some problems during the transfer of the small-scale experiments reported here to a whole lake situation. Technical problems to expose and safely remove of thousands m2 AAPs need to be solved. Human interference also needs to be taken into consideration. Importantly, the large-scale introduction of AAPs and hence the growth of periphyton may influence the ecosystem in a profound and perhaps unpredictable way.

2)The second major flaw of the paper is the complete neglect of sediment resuspension.  It is known that resuspended sediments are a primary source of internal loading of nutrients and a major feedback of macrophytes are the anchoring of sediments.  The enclosures used in this study would stop resuspension since they were open on the top and bottom and prevented wind impacts.  Looking at the data, NTU, COD, TP and chl-a all decreased in the controls which could be attributed somewhat to the prevention of sediment resuspension by the enclosures.  In addition, the authors do not measure sediments in their analyses.  While they attribute the artificial plants as taking up an extrodanary amount of P, they neglected to show if in the controls, the P sedimented out from dead phytoplankton. In addition, there mechanism does not include sediments as a primary factor in these systems.  This is incorrect and anyone who is studying shallow lakes must account for sediment processes.  Please read all of the work Martin Scheffer has done on this subject including his book on shallow lake ecology.  Since the authors did not measure sediments in this study, they must discuss that as a possible mechanism throughout the text to apply these findings to any shallow system.

Thank you for your advice. We admitted the view of resuspended sediments are a primary source of internal loading of nutrients, while in this experiment, we supposed that four enclosures were open to the air and above and sediment of same circumstance thus the similar influence on that, so we did not measure the indices in sediments. Also, due to the limitation of enclosures’ area, sampling of sediment may cause disturbance. But if possible, we would measure the anchoring of sediments in the successive large-scale experiments. Additionally, the macrophytes were transplanted after 60 days, which did not cause influence on the internal loading of nutrients in the enclosures during the operational time.  

3)    If this technology is going to be applied to other systems, a breakdown of cost needs to be included.  There is no other way to need this systems except in management situations.  As a result, cost must be discussed and hopefully described somewhere in the text.

In the Lines 72-77:，we give the introduction about the cost, service life and structure of the technology. In addition, the detailed description was introduced in the reference 21, 22.

Minor comments:

Lines 1-4:  The title must be changed because in no way do the authors proved that this mechanism would work in an entire lake system.  I would change “in a shallow eutrophic lake” to “shallow waters”. 

We agree with the reviewer’s opinion on the basis of current design and give the relevant adaptation in the manuscript.

Lines 34-35: the role of macrohpytes to anchor sediments needs to be included from the onset.

Thanks for the advice. The role of aquatic macrophytes to enhance the retention of phosphorus and its corresponding reference (Scheffer Marten. 1998. Ecology of shallow lakes. London: Chapman and Hall.) have been added in the manuscript.

Line 68: “previous studies” are mentioned but neither cited or explained.  You can’t just justify something as “previous studies” you need to show the reader how you can say that statement.

In the lines 66-72 “previous studies” are cited from ‘Huang, L.C. Ecological restoration of eutrophic lake with artificial grass-take Dianchi Lake for example. Master Thesis. Institute of Hydrobiology, Chinese Acadeny of Sciences, Wuhan, Hubei, China. 2016’.

Line 91: at 4.4 m mean depth, is this a shallow lake?  Seems deeper than a shallow lake.  Again, I do not think the results from this study can be extrapolated to the rest of the lake.  You do not have data to show that.

A table needs to be included giving more detail of the lake conditions.  Do not send the reader to a paper.  The entire paper is about the changes in lake conditions so please give the reader starting conditions.  N, P, secchi, chl-a, z-max, and anything else needs to be included.  For a paper based on fixing the lake (even in the title), little is provided about the lake itself.

Dianchi Lake has a water surface of 300 km2, watershed area of 2920 km2, average depth of 4.4 m and an altitude of 1886.5 m above sea level. The experimental enclosures were built in Caohai Bay where is a small part of the whole Dianchi, which located in the north of Lake Dianchi. And average depth in the test area is is approximately 2.5m. We have seriously taken the extrapolation of the experiment’s results into consideration due to we conducted in the enclosures of Caohai and given the adjustment in the text.

Line 98:  Figure 1 is terrible.  I cannot make out what is the lake and what is the watershed.  Where were the enclosures?  Are the words, study site by the mesocosm site or is that label for the entire map?  The right and bottom inset is not helpful as tall.  Finally, the Figure description is too basic.  Please help the reader see what is needed here.

We want to convey the information that study site Caohai Bay is located in the north of Dianchi Lake (24°51′N, 102°42′E, Fig. 1) through Figure 1. According to the editor’s suggestion that the map should show the location of the lake within China. We have changed the map into clear version.

Lines: 158-172: the measurement of bacteria is a surprise to the reader.  It needs to be stated what is the question and use of this data.

Line 85-88 we give the explanation that one of main objectives of this study was the investigation of the dynamics of periphyton attached on artificial substrates during the experiment.

Line 291-295 The explanation of “Dynamics of bacterial communities with the development of periphyton biofilms attached to AAPs” is “The structure and succession of the bacterial communities were compared and analyzed during the intervention of the AAPs, which can be regarded as an artificial micro-ecosystem in which many microorganisms, including new prokaryotic and eukaryotic colonizers, interact in the periphyton biofilms. Gene amplicon sequencing data of samples CW, TW and TB were acquired based on the 16S rRNA.”

Lines191-213: Again, a table is needed here.  There is no way a reader can truly get the information among the statements and “respectfully’s”. 

We have summarized the results of the average removal efficiency of nutrients with different treatments are summarized in Table below. While we suppose that the Line graph may be more  intuitive and directive for readers, so we recommend reservation.

Initial water quality index of the experimental enclosures and removal efficiency of nutrients with different treatments after two months’ operation (means ± SE).

Line 213: stating that this can be applied to all eutrophic waters is an overstatement.  Maybe, shallow littoral areas or small eutrophic ponds such as agricultural impoundments or aquaculture ponds.

We accept your advice and make adjustments.

Lines 261-263: The figure description needs to be expanded.  What is CW0, CW1 or TB1, TB2. 

Explanation was exhibited in lines 239-244 and lines 265-267.

CW--water samples from control enclosures without AAPs

TW--water samples from treatment enclosures within AAPs

TB--microfiber samples containing periphyton biofilm attached to AAPs

Phase 1 (the initial state): [CW0] [TW0];

Phase 2 (30 days later): [CW1] [TW1] [TB1];

Phase 3 (60 days later): [CW2] [TW2] [TB2].

Lines 314-320:  What if diversity increased in the sediments of the control?  This is not measured and is a viable place for microbial processes. Line 365: Sediments need to be included in the conceptual figure.

Thank you for your advice. While in this experiment, we mainly concerned about the dynamics of microbial community in the water column and periphyton biofilms with neglecting the similar influence on the four enclosures of same circumstance.

Line 343: change didn’t to did not

We accept.

Line 372: again, eutrophicated lakes is an over statement for the data provided.  Change this to a more similar application (shallow littoral areas or small eutrophic ponds such as agricultural impoundments or aquaculture ponds)

We have made appropriate adjustment.

Author Response File: Author Response.docx

Reviewer 2 Report

Review of “Reducing the phytoplankton biomass to promote the growth of submerged macrophytes by introducing artificial aquatic plants in a shallow lake”.

            The authors were interested in testing the idea that the introduction of artificial aquatic plants into the shallow waters of a lake would promote the growth of periphyton biofilms which in turn would remove nutrients from the water which in turn would reduce the concentrations of phytoplankton algae and in turn increase Secchi dick transparency.  They hypothesized that this process would increase the propagation of submerged aquatic plants in the lake.  They installed four enclosures in Dianchi Lake.  Two were used as controls and two were used for the experimental introduction of artificial plants.  They found that over a 60 day period the enclosures with artificial plants showed greater decreases in turbidity, total phosphorus, total nitrogen, and cell densities of phytoplankton algae.  A transplanted macrophyte (Elodea nuttallii) introduced at Day 60 grew in the experimental enclosure but not in the control.  They suggest that the introduction of artificial aquatic plants in shallow eutrophic lakes might help to restore macrophyte populations.

Their experiment supported their hypothesis that artificial aquatic plants could be used to reduce nutrients and phytoplankton numbers in shallow eutrophic lakes and help in the establishment of submersed aquatic plants.  This will be of interest to managers of shallow eutrophic lakes.  It should be pointed out, however, that this was a small experiment carried out once with few replications.  Much more work will be needed to recommend the wide application of this technique.  More information will be needed to estimate costs and the practicality of apply this method to while lakes.

I had some problems in following the manuscript in places due to the lack of details necessary to understand and replicate the work.  I will highlight some examples where work is needed.

Line 68

            Many readers will not be familiar with the four different substrates mentioned.  Describe them or at the least give references.

            Change result to results

Line 103

            The term strain is used in connection with the artificial aquatic plants.  This does not seem to fit the English definitions.  Perhaps you might want to use a different word like strand throughout the manuscript.  You might say “An AAP strand was composed…”

            More information is needed on your AAP.  Did you make them your selves?  If so, describe how you made them. If you purchased them give references that would describe them in some detail.

Line 120

            What was the sampling frequency?

Section 2.5

            I had problems in following the methods and the subsequent calculations in this section.  On Line 130 it is stated that 20 ml of water was used to extract algal cells from a 0.5 g of textile (dry weight).  How did you collect exactly 0.5 grams of the fibers?  Did you dry them before removing the algal cells?  Explain.

            What was the final volume of water that you used to remove the algal cells from the 0.5 g of microfibers?  I need to know this in order to follow Equations 1 and 2.    How does this relate to the value of V₁?  In Line 155 this is defined as the algal fluid volume collected from the microfibers of AAP, L. Is this the volume of water used from the 0.5 g sample of microfiber?

            I have a similar problem with A₂.  In Line 150 it is defined as the content of Chl a adsorbed by microfibers of AAP, µg/L.  What microfibers are you referring to?  Are they the microfibers in the 0.5 g sample or do they refer to the microfibers for the entire strand?  Shouldn’t the content be in units of µg?  How do you get µg/L.

            It would seem to me that the equations should include the value for the dry weight of fibers in the sample (0.5g) as well as the value for the volume of water used to extract the algal cells.

            It would be a great help if you included a table with the definition and units for each of the variables used in Equations 1, 2, and 3.

Figure 3

            Increase the font sizes used in this and the other figures.

Figure 3 legend

            You are plotting measured values like TP, TN, etc. and not variations.  I would remove the word variations from all your figure legends.

Line 248

            Fix the English in this statement.

Line 258

            What is meant by “communicate and protect each other”?

Figure 4.

            What are TB1 and TB2?

            I do not understand this figure.  What is the line?

Line 368

            I think you mean concentrations and not loads.

Line 369

Author Response

Review of “Reducing the phytoplankton biomass to promote the growth of submerged macrophytes by introducing artificial aquatic plants in a shallow lake”.

The authors were interested in testing the idea that the introduction of artificial aquatic plants into the shallow waters of a lake would promote the growth of periphyton biofilms which in turn would remove nutrients from the water which in turn would reduce the concentrations of phytoplankton algae and in turn increase Secchi dick transparency.  They hypothesized that this process would increase the propagation of submerged aquatic plants in the lake.  They installed four enclosures in Dianchi Lake.  Two were used as controls and two were used for the experimental introduction of artificial plants.  They found that over a 60 day period the enclosures with artificial plants showed greater decreases in turbidity, total phosphorus, total nitrogen, and cell densities of phytoplankton algae.  A transplanted macrophyte (Elodea nuttallii) introduced at Day 60 grew in the experimental enclosure but not in the control.  They suggest that the introduction of artificial aquatic plants in shallow eutrophic lakes might help to restore macrophyte populations.

Their experiment supported their hypothesis that artificial aquatic plants could be used to reduce nutrients and phytoplankton numbers in shallow eutrophic lakes and help in the establishment of submersed aquatic plants.  This will be of interest to managers of shallow eutrophic lakes.  It should be pointed out, however, that this was a small experiment carried out once with few replications.  Much more work will be needed to recommend the wide application of this technique.  More information will be needed to estimate costs and the practicality of apply this method to while lakes.

I had some problems in following the manuscript in places due to the lack of details necessary to understand and replicate the work.  I will highlight some examples where work is needed.

Line 68   Many readers will not be familiar with the four different substrates mentioned.  Describe them or at the least give references.

Thank you for your good advice.

In the line 72, which cited from  [21] ‘Huang, L.C. Ecological restoration of eutrophic lake with artificial grass-take Dianchi Lake for example. Master Thesis. Institute of Hydrobiology, Chinese Acadeny of Sciences, Wuhan, Hubei, China. 2016’.

The artificial aquatic plants (AAPs) was chosen as the most efficient for improving water quality among four different substrates (aquamats, biocompatibility carbon fiber, eco-carbon fiber and AAP) based on our previous studies. The result demonstrated that both biocompatibility carbon fiber and AAP could improve water quality by reducing the concentration of nutrients of experiment water, restraining the reproduction of algae in the water column. The removal rates of TN, TP and Chl-a by AAP and biocompatibility carbon fiber were: 66.81%, 61.85%; 57.89%, 8.26%; 88.84%, 94.03% respectively [21].

Change result to results

We accepted.

Line 103  The term strain is used in connection with the artificial aquatic plants.  This does not seem to fit the English definitions.  Perhaps you might want to use a different word like strand throughout the manuscript.  You might say “An AAP strand was composed…”

More information is needed on your AAP. Did you make them your selves?  If so, describe how you made them. If you purchased them give references that would describe them in some detail.

In the Lines 72-77: we give the introduction about the cost, service life and structure of the technology. In addition, the detailed description was introduced in the reference 21. We purchased the AAP for the corporation. A strain AAP was composed of thousands of polypropylene microfibers, which imitate the structure of natural aquatic plants. A uniform weight was used for cement balls tied at the bottom of every strain of AAP to maintain a vertical state and set a density of 1 strains/m3 in the treatment enclosures.

Line 120  What was the sampling frequency?

In the lines116-199.

Section 2.5

I had problems in following the methods and the subsequent calculations in this section.  On Line 130 it is stated that 20 ml of water was used to extract algal cells from a 0.5 g of textile (dry weight).  How did you collect exactly 0.5 grams of the fibers?  Did you dry them before removing the algal cells?  Explain.

We collect the textile through ratio of wet and dry, and weighted three times for each sample.

What was the final volume of water that you used to remove the algal cells from the 0.5 g of microfibers?  I need to know this in order to follow Equations 1 and 2.    How does this relate to the value of V1?  In Line 155 this is defined as the algal fluid volume collected from the microfibers of AAP, L. Is this the volume of water used from the 0.5 g sample of microfiber?

20 mL of a sterile saline solution was added to 0.5 g textile (dry weight) in 50 mL centrifugation tubes. After centrifuging for 2 minutes sat maximum speed, the sample was shaken horizontally at 200 rpm for 30 minutes at room temperature. This process was repeated three times and the algal fluid volume was collected. Equally, V1 is the algal fluid volume collected from the microfibers of AAP, L

I have a similar problem with A2.  In Line 150 it is defined as the content of Chl a adsorbed by microfibers of AAP, µg/L.  What microfibers are you referring to?  Are they the microfibers in the 0.5 g sample or do they refer to the microfibers for the entire strand?  Shouldn’t the content be in units of µg?  How do you get µg/L.

A1 is the content of Chl a adsorbed by all strains of AAPs (72strains) in each enclosure, μg/L, A2 is the content of Chl a adsorbed by microfibers of AAP ( 0.5 g sample), μg/L. The Chl a was measured by spectrophotometry.

It would seem to me that the equations should include the value for the dry weight of fibers in the sample (0.5g) as well as the value for the volume of water used to extract the algal cells.

It would be a great help if you included a table with the definition and units for each of the variables used in Equations 1, 2, and 3.

Thank you for your good advice. M₁ is the dry weight of microfibers of AAP, g; V₁ is the algal fluid volume collected from the microfibers of AAP, L.

Figure 3  Increase the font sizes used in this and the other figures.

Accept.

Figure 3 legend

You are plotting measured values like TP, TN, etc. and not variations.  I would remove the word variations from all your figure legends.

Accept.

Line 248  Fix the English in this statement.

Accept.

Line 258  What is meant by “communicate and protect each other”?

I mean,pioneer planktonic algal cells attached to the substrata and biological adhesion continued to form micro-colonies, which helped them to defend adverse conditions and protect each other.

Figure 4.

What are TB1 and TB2?

I do not understand this figure.  What is the line?

Explanation was exhibited in lines 239-244 and lines 265-267.

TB--microfiber samples containing periphyton biofilm attached to AAPs

Phase 2 (30 days later): [TB1];

Phase 3 (60 days later): [TB2].

Line 368

I think you mean concentrations and not loads.

We accept.

Author Response File: Author Response.docx

Reviewer 3 Report

Reducing the phytoplankton biomass to promote  the growth of submerged macrophytes by introducting artificial aquatic palnts in a shallow eutrophic lake

The article concerns the use of artificial aquatic plants to improve water quality in a highly eutrophic reservoir. The authors present here the results of experimental studies carried out in shallow Dianchi Lake. The methods of the experiments, the characteristics of the stydy area and the description of the results obtained are presented in detail. A few detailed comments are given below:

Line 68 - what does APP abbreviation mean?

Line 90-92 - add more morphometric parameters of the Dianchi Lake: maximum depth, length, width of the lake, volume of water. Describe in more detail the use of the lake catchment area and the bay where the experiments were conducted. Moreover, there is no precise information about the water quality of the lake and the bay: N and P content, chlorophyll concentration, etc.

Fig.1: Less visible differences in the thickness of the line showing the border between Dianchi Basin and Lake Dianchi

Fig.1 . There is no description of the red star in the legend. Bathymetric map is recommended

Line 100-106

How far away were the enclosures from each other?

At what time of year (month) were experiments carried out?

The length of the experiment was given in the abstract, but there is no such information in the Materials and Methods chapter.

Line 128-157

Was the species composition of all phytoplankton groups determined?

Figure 3 - low visibility of signatures on the X-axis; What does the point on the graph mean (is it the average of several samples?)?

Line 223 - no unit

Line 229 - How did you identify adsorption and absorption during the experiment?

Line 236-259 - which phytoplankton species dominated in CW and which in TW and TB? Which species of Cyanobacteria showed the highest share and abundance? Were the periphyton species also found in water?

Line 270-283

Describe what mechanisms and factors caused such reconstruction of the phytoplankton species structure.

Fig.5 Incomplete description of the figure

Table 1 - Were the average values from the whole experiment used in the calculations?

Line 339-363

A comparison with other studies aimed at assessing the effects of such a restoration method would be useful.

To what depth of water can this method be used?

Did the ratio of TN to TP change during the experiment?

How could such a restoration method look like in practice for the whole lake?

The hydrobiological literature uses the term Cyanobacteria, Chlorophyceae, Bacillariophyceae.

Author Response

Comments 3

Reducing the phytoplankton biomass to promote  the growth of submerged macrophytes by introducting artificial aquatic palnts in a shallow eutrophic lake

The article concerns the use of artificial aquatic plants to improve water quality in a highly eutrophic reservoir. The authors present here the results of experimental studies carried out in shallow Dianchi Lake. The methods of the experiments, the characteristics of the stydy area and the description of the results obtained are presented in detail. A few detailed comments are given below:

Line 68 - what does APP abbreviation mean?

Thank you for your good advice.

We guess that the reviewer means AAP,  we give the explanation that AAP abbreviation means artificial aquatic plant in the line 13.

Line 90-92 - add morphometric parametermore morphometric parameters of the Dianchi Lake: maximum depth, length, width of the lake, volume of water. Describe in more detail the use of the lake catchment area and the bay where the experiments were conducted. Moreover, there is no precise information about the water quality of the lake and the bay: N and P content, chlorophyll concentration, etc.

Fig.1: Less visible differences in the thickness of the line showing the border between Dianchi Basin and Lake Dianchi

Fig.1 . There is no description of the red star in the legend. Bathymetric map is recommended

Dianchi Lake has a water surface of 300 km², watershed area of 2920 km², average depth of 4.4 m and an altitude of 1886.5 m above sea level. The experimental enclosures were built in Caohai Bay where is a small part of the whole Dianchi, which located in the north of Lake Dianchi. And average depth in the test area is is approximately 2.5m.

We have summarized the results in Table below. While we suppose that the Line graph may be more intuitive and directive for readers, so we recommend reservation.

We have changed the map into clear version.

Line 100-106 How far away were the enclosures from each other?

Every enclosure is next to each other.

At what time of year (month) were experiments carried out?

January to April.

The length of the experiment was given in the abstract, but there is no such information in the Materials and Methods chapter.

Thank you for your suggestion, besides, we gave the length of the experiment in the figures of results and discussion.

Line 128-157

Was the species composition of all phytoplankton groups determined?

The species of all phytoplankton were identified by microscope and were exhibited by showing phylum.

Figure 3 - low visibility of signatures on the X-axis; What does the point on the graph mean (is it the average of several samples?)? Line 223 - no unit

The point on the graph mean 15^th and 45^th day,  the unit was exhibited in the Y-axis.

Line 229 - How did you identify adsorption and absorption during the experiment?

We suppose that phytoplankton and planktonic bacteria in the water column were physically adsorbed in AAP and nutrients were absorbed biologically by periphyton biofilms. 

Line 236-259 - which phytoplankton species dominated in CW and which in TW and TB? Which species of Cyanobacteria showed the highest share and abundance? Were the periphyton species also found in water?

In CW, Microcystis sp. dominated in CW; Scenedesmus sp., Pseudanabaena sp., Microcystis sp. dominated in TW; in the lines 283-285: An interesting result is that the ratios of Bacillariophyta in periphyton biofilms attached to artificial aquatic plants (TB) accounted for a mean of 48.5%, and the dominant species were Achnanthes exigua, Gomphonema acuminatum, Cymbella pusilla, Navicula placentula and Navicula graciloides.

Describe what mechanisms and factors caused such reconstruction of the phytoplankton species structure.

When an AAP with a large specific surface area was introduced into the eutrophic lake water, large amounts of nutrients (e.g., N, P and COD), phytoplankton and planktonic bacteria in the water column would be rapidly adsorbed in thousands of microfibers in AAP to form the periphyton biofilm. Nutrients could then be gradually assimilated by adherent micro-algae, and biodegraded by attached bacteria with the amplification of periphyton biofilms. Thus, the reduced availability of nutrients and the competition between the planktonic and attached microbial community would lead to the restraint of cyanobacterial growth and the subsequent change in planktonic and attached microbial communities.

Fig.5 Incomplete description of the figure

Have adjusted.

Table 1 - Were the average values from the whole experiment used in the calculations?

Average values of each three samples.

Line 339-363 A comparison with other studies aimed at assessing the effects of such a restoration method would be useful. To what depth of water can this method be used? How could such a restoration method look like in practice for the whole lake?

In the successive design, we hope to investigate a large-scale experiment to explore the feasibility of periphyton biofilms attached on AAPs to reduce cyanobacteria blooms in a suitable lake. And at the end of the manuscript, we would give discussion about limitations and applications of this idea. Such as, we envision to encounter some problems during the transfer of the small-scale experiments reported here to a whole lake situation. Technical problems to expose and safely remove of thousands m2 AAPs need to be solved. Human interference also needs to be taken into consideration. Importantly, the large-scale introduction of AAPs and hence the growth of periphyton may influence the ecosystem in a profound and perhaps unpredictable way.

Did the ratio of TN to TP change during the experiment?

According to the calculated result, the ratio of TN to TP keep increased during the experiment in the control enclosures while that of the experimental enclosures rise earlier and then fall.

The hydrobiological literature uses the term Cyanobacteria, Chlorophyceae, Bacillariophyceae.

Thank you for your advice, while we are referring to phylum-level algae , so we suggest that Cyanophyta, Chlorophyta and Bacillariophyta may be more appropriate

Author Response File: Author Response.docx

Round 2

Reviewer 2 Report

Some of my comments were answered in the response to the review but were not answered in the text.  It is not sufficient to tell me the answers.  The reader of the paper will want the information requested.  Please use the text to respond to the following comments.

Line 106

            The term strain is used in connection with the artificial aquatic plants.  This does not seem to fit the English definitions. 

Define what you mean by a strain, or perhaps you might want to use a different word like strand throughout the manuscript.  You might say “An AAP strand was composed…”

            More information is needed on your AAP.  Did you make them your selves?  If so, describe how you made them. If you purchased them give references that would describe them in some detail.  Your thesis is not going to be widely available to limnologists in other countries who might want to repeat your experiments.

Line 121

            What was the sampling frequency?  How often were samples taken?

Section 2.5

            I had problems in following the methods and the subsequent calculations in this section.  On Line 130 it is stated that 20 ml of water was used to extract algal cells from a 0.5 g of textile (dry weight).  How did you collect exactly 0.5 grams of the fibers?  Did you dry them before removing the algal cells?  Explain.

            What was the final volume of water that you used to remove the algal cells from the 0.5 g of microfibers?  I need to know this in order to follow Equations 1 and 2.    How does this relate to the value of V₁?  In Line 155 this is defined as the algal fluid volume collected from the microfibers of AAP, L. Is this the volume of water used from the 0.5 g sample of microfiber?

            I have a similar problem with A₂.  In Line 150 it is defined as the content of Chl a adsorbed by microfibers of AAP, µg/L.  What microfibers are you referring to?  Are they the microfibers in the 0.5 g sample or do they refer to the microfibers for the entire strand?  Shouldn’t the content be in units of µg?  How do you get µg/L.

            It would seem to me that the equations should include the value for the dry weight of fibers in the sample (0.5g) as well as the value for the volume of water used to extract the algal cells.

            It would be a great help if you included a table with the definition and units for each of the variables used in Equations 1, 2, and 3.

Figure 3

            Increase the font sizes used in this and the other figures.

Line 251

The verb should be “were inhibited”

the phytoplankton were inhibited

Lines 258-259

            What is meant by “communicate and protect each other”?

Figure 4.

            Explanations are needed in text or legend

            I do not understand this figure.  What is the line?  What are the bars?

Line 373

            Not sure on the use of the word incremental

            I would replace “incremental of” to “increases in”

Author Response

Thank you for your advice.

Line 106

The term strain is used in connection with the artificial aquatic plants.  This does not seem to fit the English definitions.

Define what you mean by a strain, or perhaps you might want to use a different word like strand throughout the manuscript.  You might say “An AAP strand was composed…”

More information is needed on your AAP.  Did you make them your selves?  If so, describe how you made them. If you purchased them give references that would describe them in some detail.  Your thesis is not going to be widely available to limnologists in other countries who might want to repeat your experiments.

We accepted and adjusted in the text. The artificial aquatic plant (AAP) was purchased from a Chinese company (Wuxi Pengbo Environmental Protection Technology Co., LTD). In the lines 72-77, we add more details about AAP in the reference 22.

Line 121

What was the sampling frequency?  How often were samples taken?

In section 2.3, lines 118-121: For eliminating spatial heterogeneity, the water samples were collected at 3 sampling sites horizontally in the same enclosure and twice a month. Similarly, the microfiber samples were carefully cut from 3 different AAP strands in the same enclosure with sterile scalpels and then weighed for microbial analyses once per month.

Section 2.5

I had problems in following the methods and the subsequent calculations in this section.  On Line 130 it is stated that 20 ml of water was used to extract algal cells from a 0.5 g of textile (dry weight).  How did you collect exactly 0.5 grams of the fibers?  Did you dry them before removing the algal cells?  Explain.

Sorry for not describing clearly in text. We collect enough fresh textile sample, then weight the sample (wet weight) equal to 0.5g dry weight( calculated by the ratio of wet and dry ), and weighted three times for each sample.

 What was the final volume of water that you used to remove the algal cells from the 0.5 g of microfibers?  I need to know this in order to follow Equations 1 and 2.    How does this relate to the value of V1?  In Line 155 this is defined as the algal fluid volume collected from the microfibers of AAP, L. Is this the volume of water used from the 0.5 g sample of microfiber?

In the lines 132-138: 20 mL of a sterile saline solution was added to 0.5 g textile (dry weight) in 50 mL centrifugation tubes. After centrifuging for 2 minutes at maximum speed, the sample was shaken horizontally at 200 rpm for 30 minutes at room temperature. This process was repeated three times and the algal fluid volume was collected. Equally, V1 is the total algal fluid volume collected from the microfibers of AAP, L

I have a similar problem with A2.  In Line 150 it is defined as the content of Chl a adsorbed by microfibers of AAP, µg/L.  What microfibers are you referring to?  Are they the microfibers in the 0.5 g sample or do they refer to the microfibers for the entire strand?  Shouldn’t the content be in units of µg?  How do you get µg/L.

A1 is the content of Chl a adsorbed by all strains of AAPs (72strains) in each enclosure with the volume of 72000 L, μg/L; A2 is the content of Chl a adsorbed by microfibers of AAP ( 0.5 g sample), μg/L. The Chl a was measured by spectrophotometry.

It would seem to me that the equations should include the value for the dry weight of fibers in the sample (0.5g) as well as the value for the volume of water used to extract the algal cells.

It would be a great help if you included a table with the definition and units for each of the variables used in Equations 1, 2, and 3.

Thank you for your good advice while we suppose that the explanation of Equations 1, 2, and 3 is detailed in the text.

A₁ =                                 (1)

B₁ =                                 (2)

C₁ =                                           (3)

A₁ ─ the content of Chl a adsorbed by all strands of AAPs in each enclosure, μg/L;

A₂ ─ the content of Chl a adsorbed by quantitative microfibers of AAP, μg/L;

B₁ ─ the cell density of attached algae by all strands of AAPs in each enclosure, cells/L;

B₂ ─ the cell density of attached algae by quantitative microfibers of AAP, cells/L;

C₁ ─ the TP content adsorbed by periphyton biofilms attached to all strains of AAPs in each enclosure, mg/L;

C₂ ─The TP content adsorbed by periphyton biofilms attached to the quantitative microfibers of AAP, mg/g;

M₁ ─ the dry weight of quantitative microfibers of AAP, g;

M₂ ─ the average dry weight of per strain of AAP, g;

V₁ ─ the algae fluid volume collected from quantitative microfibers of AAP, L;

72 ─ the number of AAPs of each enclosure, strain;

72000 ─ the volume of each enclosure, L.

Figure 3  Increase the font sizes used in this and the other figures.

The format of Figure 3 meets the requirements of Water, which the quality of Figure 3 exceeds 300 dpi.

Line 251  The verb should be “were inhibited”

the phytoplankton were inhibited

We accepted and adjusted in the text .

Lines 258-259  What is meant by “communicate and protect each other”?

From the view of adsorption, pioneer planktonic algal cells attached to the substrata and biological adhesion continued to form micro-colonies (Watnick et al., 2000). The attachment of micro-algal cells to a stable surface increased their existence relative to those in the water column (Hickman et al., 2005), which made them communicate and protect each other to adapt environmental conditions.

Figure 4.  Explanations are needed in text or legend

I do not understand this figure.  What is the line?  What are the bars?

We have given detailed explanation in the lines 241-246 of the text and legend (lines 267-269).

Bars means the cell density, line means the concentration of Chl a.

Line 373  Not sure on the use of the word incremental

I would replace “incremental of” to “increases in”

We accepted and adjusted in the text .

Author Response File: Author Response.docx