Microalgal Growth in Aquaculture Effluent: Coupling Biomass Valorisation with Nutrients Removal

Round 1

Reviewer 1 Report

 

In the present manuscript, the authors investigate the cultivation of a selected microalgal species in a wastewater/effluent medium, obtained from an aquaculture trout facility. The main objective is to replace a commonly used chemically defined medium with this wastewater stream and simultaneously remove the N and P sources from the effluent for they are environmentally hazardous (as eutrophication factors). In addition, the authors state that the produced microalgal biomass can be utilized as fish feed in an aquaculture farm.

In general, the manuscript is clearly written and easy to follow. On the other hand, the number of the conducted experiments is very limited and therefore the described statistical analysis of obtained measurements is ambiguous. Thus, the overall value of the present contribution is low.

Moreover, the authors should provide satisfactory answers to the following comments/questions before the manuscript can be possibly considered for a new review and possible publication:

1. There is not any proof that the produced microalgal biomass can be used for aquaculture feed. Thus, the last paragraph of Section 3 on Results and Discussion is rather a feature objective than a proof of the concept.

2. The applied illumination profile is not clear. Did the authors used night/day periods?

3. Why did the authors use different N and P concentrations for the experiments in AW and C+ assays (there is at least a 2-fold increase in N and 5-fold increase in P in the C+ runs in comparison to the AW experiments)? For comparison reasons, they should have carried out experiments under similar N and P concentrations.

4. There is no information regarding the carbon CO₂ source. Can be assumed that the CO₂ source was that in the supplied air? The authors should provide some details regarding the CO₂ concentration and comment accordingly on its effect.

5. In Table 3, row 4, the reported value for λ is equal to zero. Is this correct? Moreover, in the first and fourth rows of Table 3, the calculated confidence intervals for λ are very large, which means that the value of λ can be equal to zero or/and negative. This is a major shortcoming of the present manuscript due to the very limited number of experiments used in the statistical analysis.

6. The abstract should be rewritten to reflect the actual contribution of the present work.

7. Page 4, line 135: The authors include a negative control test without the addition of microalgae. What is the reason for this control experiment?

8. Page 4, line 137: Explain why the pH of the culture medium had to be regulated at exactly 7.53? Is this an optimal value for the microalgae growth? Please provide experimental evidence and results.

9. Page 4, line 160: Please explain how does the lyophilization of the collected biomass ensures cell rupture. Provide experimental evidence and results.

10. How was the biomass concentration measured? Did the authors use the calibration curve described in page 4, line 172 or they employed another method? Describe clearly the experimental procedure followed to obtain the calibration curve. Report the calculated calibration equation and its application limits.

11. Page 9, lines 321-323: Despite that nitrogen limiting conditions applied to the AW experiments, for at least 6-7 hours (see Figure 3), no lipids enhancement was measured according to the results shown in Table 5 in comparison to the C+ experiments. Please justify this observation.

 

Author Response

Response to Reviewer 1 Comments

 

In the present manuscript, the authors investigate the cultivation of a selected microalgal species in a wastewater/effluent medium, obtained from an aquaculture trout facility. The main objective is to replace a commonly used chemically defined medium with this wastewater stream and simultaneously remove the N and P sources from the effluent for they are environmentally hazardous (as eutrophication factors). In addition, the authors state that the produced microalgal biomass can be utilized as fish feed in an aquaculture farm.

 

In general, the manuscript is clearly written and easy to follow. On the other hand, the number of the conducted experiments is very limited and therefore the described statistical analysis of obtained measurements is ambiguous. Thus, the overall value of the present contribution is low.

 

Moreover, the authors should provide satisfactory answers to the following comments/questions before the manuscript can be possibly considered for a new review and possible publication

 

First, we would like to thank you for your valuable comments and suggestions, which helped us to improve the manuscript. Below we try to address all the points, which you have indicated in your revision. The new/changed text is tracked in the manuscript.

 

Point 1: There is not any proof that the produced microalgal biomass can be used for aquaculture feed. Thus, the last paragraph of Section 3 on Results and Discussion is rather a feature objective than a proof of the concept.

Response 1: Since we were not able to conclude whether it would be possible to use the produced biomass as feed for aquaculture we change the goal and the title of the article to more suitable ones. The goal of this study was to use microalgae to treat aquaculture wastewater and then valorise the produced biomass through the analysis of the biochemical composition of the biomass. See lines 20-26, page 1; lines 108-111, page 3 and lines 400-403, page 11.

In the last paragraph of Section 3, different studies are presented that explain how microalgae can be applied as a supplement to the nutritional diet used in aquaculture, as an example of biomass valorisation.

 

Point 2: The applied illumination profile is not clear. Did the authors used night/day periods?

Response 2: The light was continuously supplied by LEDs, that is, the light:dark ratio was 24:0. The sentence was changed in the manuscript to make it clearer, as we understood the confusion. Please see lines 121 page 3 and line 168 page 4.

 

Point 3: Why did the authors use different N and P concentrations for the experiments in AW and C+ assays (there is at least a 2-fold increase in N and 5-fold increase in P in the C+ runs in comparison to the AW experiments)? For comparison reasons, they should have carried out experiments under similar N and P concentrations.

Response 3: As positive control (C+) is it is common to use optimal growth conditions, hence the use of the modified OECD medium. To compare the growth in wastewater versus growth in a medium where conditions are optimal.

 

Point 4: There is no information regarding the carbon CO₂ source. Can be assumed that the CO₂ source was that in the supplied air? The authors should provide some details regarding the CO₂ concentration and comment accordingly on its effect.

Response 4: The CO₂ supplied corresponds to the CO₂ concentration in the air 400 ppm. The CO₂ was provided through the injection of atmospheric air using the air pumps (this information was added to the text, see lines 171-172, page 4). Given the concentration of CO₂ in the air, the microalgal growth was limited by this concentration. Moreover, this concentration did not affect the ph of the culture.

 

Point 5: In Table 3, row 4, the reported value for λ is equal to zero. Is this correct? Moreover, in the first and fourth rows of Table 3, the calculated confidence intervals for λ are very large, which means that the value of λ can be equal to zero or/and negative. This is a major shortcoming of the present manuscript due to the very limited number of experiments used in the statistical analysis.

Response 5: The suggestion of the reviewer was considered and analysed. The Gompertz model parameters were determined, minimising the sum of squared errors between the experimental and model data using Microsoft Excel Solver. For this optimisation, some constraints were added for some parameters: for instance, λ should be greater or equal to zero. As this model is generated with achieved experimental data, this parameter was considered zero due to this constraint. Consequently, the calculated confidence interval was larger.

 

Point 6: The abstract should be rewritten to reflect the actual contribution of the present work.

Response 6: The goal of this study was to use microalgae to treat aquaculture wastewater and then valorise the produced biomass through the analysis of the biochemical composition of the biomass. It is important to look at microalgae from a biorefinery point of view and try to take advantage of all the benefits that microalgae have. Thus, we try to combine their great capacity to treat wastewater and take advantage of the produced biomass, analysing its composition for subsequent valorisation. The abstract was rewritten to better represent the goal of the presented work.

 

Point 7: Page 4, line 135: The authors include a negative control test without the addition of microalgae. What is the reason for this control experiment?

Response 7: The negative control is used to evaluate the effect of adding microalgae to the treatment. If in the negative control test the nutrients decrease (due to the presence of microorganisms) as much as in the microalgae tests, their addition is not necessary.

 

Point 8:  Page 4, line 137: Explain why the pH of the culture medium had to be regulated at exactly 7.53? Is this an optimal value for the microalgae growth? Please provide experimental evidence and results.

Response 8: To ensure that the experimental conditions were similar, the pH of the microalgal cultures was adjusted daily to match the value of the negative control.

 

Point 9: Page 4, line 160: Please explain how does the lyophilization of the collected biomass ensures cell rupture. Provide experimental evidence and results.

Response 9: We corrected the phrase in question in the manuscript, see lines 182-185, page 5.

 

Point 10: How was the biomass concentration measured? Did the authors use the calibration curve described in page 4, line 172 or they employed another method? Describe clearly the experimental procedure followed to obtain the calibration curve. Report the calculated calibration equation and its application limits.

Response 10: The biomass concentration was measured using the calibration curve described in page 4, line 172. To obtain the calibration curve, a known volume of microalgal culture at different concentrations was vacuum filtered with 0.45 µm cellulose acetate membranes previously dried at 60 °C for 24 h. Then, after the filtration, the membranes were dried at 105 °C for 24 h. The difference between the membrane masses divided by the filtered volume corresponds to the biomass concentration in terms of dry weight. Each microalgal culture concentration was evaluated in terms of optical density at 680 nm (OD₆₈₀). And then, a linear regression between OD₆₈₀ and biomass concentration was established to obtain the calibration curve. The reference of the described method and the calibration curve were added to the text, see lines 195-197, page 5.

 

Point 11:  Page 9, lines 321-323: Despite that nitrogen limiting conditions applied to the AW experiments, for at least 6-7 hours (see Figure 3), no lipids enhancement was measured according to the results shown in Table 5 in comparison to the C+ experiments. Please justify this observation.

Response 11: Normally, microalgae accumulate carbohydrates as starch in cells. They use this compound as a source of energy. When stressed, microalgae can accumulate compounds that will serve as a source of energy such as carbohydrates and lipids. As shown in Table 5, although the lipid content was the same in the two experiments, the carbohydrate content was higher in AW experiments, revealing a stress response to nitrogen-limiting conditions.

 

Reviewer 2 Report

In this study, the authors evaluated the growth of Chlorella vulgaris in aquaculture wastewater and possible valorisation of the produced biomass as feed for aquaculture. In general, the manuscript is well written. The novelty of the presented research is poorly supported. Overall, I recommend the manuscript for publication after revision.

 Comments

The novelty of the presented research is poorly supported. Many papers on the use of aquaculture effluent have been published. What is the novelty of the present study? The aspect of novelty of the research should be clearly defined.

Author Response

Response to Reviewer 2 Comments

 

In this study, the authors evaluated the growth of Chlorella vulgaris in aquaculture wastewater and possible valorisation of the produced biomass as feed for aquaculture. In general, the manuscript is well written. The novelty of the presented research is poorly supported. Overall, I recommend the manuscript for publication after revision.

 

First, we would like to thank you for your valuable comments and suggestions, which helped us to improve the manuscript. Below we try to address all the points, which you have indicated in your revision. The new/changed text is tracked in the manuscript.

 

Point 1: The novelty of the presented research is poorly supported. Many papers on the use of aquaculture effluent have been published. What is the novelty of the present study? The aspect of novelty of the research should be clearly defined.

Response 1: The goal of this study was to use microalgae to treat aquaculture wastewater and then valorise the produced biomass through the analysis of the biochemical composition of the biomass. It is important to look at microalgae from a biorefinery point of view and try to take advantage of all the benefits that microalgae have. Thus, we try to combine their great capacity to treat wastewater and take advantage of the produced biomass, analysing its composition for subsequent valorisation. See Abstract and lines 107-109.

 

Reviewer 3 Report

Dear Authors, the reviewer´s comments are attached.

Comments for author File: Comments.pdf

Author Response

Response to Reviewer 3 Comments

 

The subject of the present manuscript is interesting and presents scientific relevance. However, I missed innovation in the present study, what is innovative about your work compared to other works conducted in this line of research. It is important to emphasize the innovative character of your work. In order for the present manuscript to be accepted, it is important to mention the novelty it presents to science. In addition, several aspects must be taken into account during the writing of the text, methodology, as well as the presentation of the results

 

First, we would like to thank you for your valuable comments and suggestions, which helped us to improve the manuscript. Below we try to address all the points, which you have indicated in your revision. The new/changed text is tracked in the manuscript.

 

Point 1: The abstract should be improved drawing attention to the novelty of the study.

Response 1: The goal of this study was to use microalgae to treat aquaculture wastewater and then valorise the produced biomass through the analysis of the biochemical composition of the biomass. It is important to look at microalgae from a biorefinery point of view and try to take advantage of all the benefits that microalgae have. Thus, we try to combine their great capacity to treat wastewater and take advantage of the produced biomass, analysing its composition for subsequent valorisation. The abstract was rewritten to better represent the goal of the presented work.

 

Point 2: Abstract: “However, the protein content obtained (17.93±1.21%) was lower than expected”. The authors should explain this behavior, but not in Abstract section.

Response 2: This phrase has been rephrased to “However, the protein content obtained (17.93±1.21%) was lower than the ones mentioned in the literature” (see Lines 32-33, page 1)

 

Point 3: The introduction should be more detailed, presenting the studies that support the research in question, as well as quantitative data from authors who have already carried out similar studies.

Response 3: The suggestion of the reviewer was considered. More studies were added to the manuscript. Please see lines 76-82 page 2.

 

Point 4: Check that all abbreviations are described in the text.

Response 4: All abbreviations are described in the text.

 

Point 5: References must be reviewed and formatted according to the journal's standards.

Response 5: All references have been reviewed and formatted according to the journal style.

 

Point 6: It is important to draw attention to the real novelty of your study and how it will contribute to the literature.

Response 6: The goal of this study was to use microalgae to treat aquaculture wastewater and then valorise the produced biomass through the analysis of the biochemical composition of the biomass. It is important to look at microalgae from a biorefinery point of view and try to take advantage of all the benefits that microalgae have. Thus, we try to combine their great capacity to treat wastewater and take advantage of the produced biomass, analysing its composition for subsequent valorisation. Please see the Abstract.

 

Point 7: Materials and methods can be detailed, including references to original methodologies.

Response 7: All materials and methods are detailed and referenced.

 

Point 8: “The biochemical analysis of the biomass revealed contents of lipids, carbohydrates, and pigment typical and similar to values reported in other studies. However, the protein content was lower than expected. Nevertheless, the compounds quantification did not allow to conclude whether it would be possible to use the biomass to supplement the nutritional diet applied in aquaculture”. Once again, the authors state that it was below expectations, but they do not present any justification

Response 8: This happened possibly due to a stress response to the nutrient depletion that occurred in the final growth stage (see Figure 3). During stress conditions, the protein content in microalgae decreases. This information was added to the manuscript.

 

 

Round 2

Reviewer 1 Report

 

In general, the revised manuscript is an improvement over its original version. The authors have answered/addressed most of the questions raised in the first review of the manuscript. However,

1. The authors did not perform any additional experiments in order to obtain statistically correct estimates of the model parameters. Specifically, in Table 3 the confidence interval for the lag time (λ) includes negative values, which does not have any physical meaning at all. This is still a major shortcoming of the revised manuscript.

2. The illumination profile was regulated to day/night ratio equal to 24/0 hrs. This practice deviates from the usual 16/8 or 14/10 ratios. No sufficient justification is provided for this choice. This means that the need for continuous illumination by artificial lighting will prevent the scaling-up of the process in an open system.

 

Author Response

Response to Reviewer 1 Comments

 

In general, the revised manuscript is an improvement over its original version. The authors have answered/addressed most of the questions raised in the first review of the manuscript. However,

Point 1: The authors did not perform any additional experiments in order to obtain statistically correct estimates of the model parameters. Specifically, in Table 3 the confidence interval for the lag time (λ) includes negative values, which does not have any physical meaning at all. This is still a major shortcoming of the revised manuscript.

Response 1: We agree with the reviewer concerning the physical meaning of negative values of lag time. We would like to clarify how these values were determined. The lag time (and other Gompertz parameters) were determined with experimental data (TN, NO₃-N and PO₄-P concentrations) achieved by the average of two samples from the three cultures (the assay was in triplicate) grown in wastewater in the same conditions (6 values for each pollutant at each time). The data from additional experiments with other conditions cannot be added to the already collected data (the parameters are specific to the tested conditions), not solving the issue pointed out by the reviewer.

As we mentioned in the first revision, we determined the model parameters, minimising the sum of squared errors with the constraint that lag time should be greater or equal to zero. This model depends on the achieved experimental data, that show a good adaptation of microalgae to the tested effluent. Consequently, the minimum value of the lag time parameter (zero) was achieved with a corresponding standard deviation. In terms of the confidence interval of this parameter, we should not consider the negative values. If this information is required, we can add it as a note below the Table 3.

 

Point 2: The illumination profile was regulated to day/night ratio equal to 24/0 hrs. This practice deviates from the usual 16/8 or 14/10 ratios. No sufficient justification is provided for this choice. This means that the need for continuous illumination by artificial lighting will prevent the scaling-up of the process in an open system. 

Response 2: We understand the concern of the reviewer. The light:dark ratios of 16:8 or 14:10 are used by several authors to simulate the natural photoperiod. However, to simulate outdoor conditions, these experiments should also consider the daily variation of light intensity and also solar radiation spectrum, which have a strong influence on microalgal growth and composition. Therefore, it is difficult to simulate real conditions on a laboratory scale/environment.

In this study, the light:dark ratio of 24:0 was used because it promotes continuous photoautotrophic growth. The scaling-up of this process can be done in a closed system with LED lights (with high energy conversion) and the required energy can be provided by renewable sources (solar, wind, and geothermal, among others). As an example, an innovative photobioreactor (PBR) was already developed in our research group with these characteristics. In the first experiments with this PBR, achieved biomass areal productivities were significantly higher than the ones achieved in the conventional designs, showing promising results for several applications of microalgal cultures.

Author Response File: Author Response.pdf

Reviewer 3 Report

Dear authors, thank you for your major revision.

Author Response

Thanks for your suggestions for the improvement of the paper.