Microalgal Systems, a Green Solution for Wastewater Conventional Pollutants Removal, Disinfection, and Reduction of Antibiotic Resistance Genes Prevalence?
and Olga C. Nunes 1,2Round 1
Reviewer 1 Report
The paper of Amaro et al deals with a really important question: how microalgae could contribute to wastewater treatment not just for chemical, but also biological point of view. The topic is important and relevant. The manuscript is quite well written, but I have concerns with some wording first appearing already in the Abstract:
P1L15-16 (and later throughout the manuscript): In my opinion, the principle to consider antibiotic resistance genes as pollutants is not correct. It suggest that these genes are present freely in wastewaters and cause similar problems as other organic pollutants, but the situation is far from this. I also don't think it is right to call coliform bacteria as pollutants. I think I understand why Authors use these terms, but some scientifically more relevant terms should be find to describe these agents.
P1L17-18 (and later throughout the manuscript): I do not agree, that the disappearance of coliform and other bacteria carrying antibiotic resistance genes because of the presence of C. vulgaris or other algae could be called “removal”, unless these algae feed on these bacteria, which is not characteristic to green algae, Chlorella among them. Even in the case of toxic or allelopathic compounds, the reduction of the proliferation of these bacteria could be the more correct term (as it is mentioned on P2L47-48).
Beside this, I feel Materials and Methods not detailed enough, and the discussions and explanations of some results are poor.
My detailed comments are the following:
Introduction
P1L31: “… natural water bodies.”
P2L45: The literature cited in the text should be numbered consecutively. So it is not correct that citation 13 is followed by citation 48.
P2L55-56: The sentence is a little bit misleading: it sounds as microalgae would generally produce humic substances. If this is the situation, there should be some more related example from the literature (e.g. from the cited ones, if they present such examples).
Materials and Methods
Nitrogen is as important for microbial growth as phosphorous. What was the reason not to measure nitrogen forms, at least inorganic ones, assuming that algae are using meanly them as nitrogen sources?
P3L102: Why continuous irradiation was used for the algal experiments? It does not represent “real life” situation.
P3L103-113: There was two C. vulgaris cultures with UWW. Why just two? There was one positive control (with sterile UWW) and one negative control (with original UWW). There should be one without the algae and with sterile UWW. What was the reason not to use such control?
To avoid dilution effect, filtered (or centrifuged) Chlorella medium should be added to controls in the same volume.
2.3 and 2.4 subchapters: The volumes of collected samples are not indicated.
Were the missing volumes removed during sampling refilled to the cultures in some form, or not? If not, could the decreasing volume affect the results? Please, explain.
2.9 subchapter: How many repetitions were carried out? The mentioned analyses of variances can be carried out only with at least three repetitions.
Results and Discussion
P7L240-243: In my opinion COD is not removed, but decreased (because chemical oxygen demand can not be accumulated or adsorbed as phosphate).
P8L278-283: It is hard to understand, how humic acid concentration connected to the presence of Chlorella vulgaris. Is there any evidence that green algae are secreting humic substances (or contribute to the presence of humic substances) in significant extent? In a recent study, C. vulgaris was present as a control (the one which less related to humic substances; Wu et al., 2022). Furthermore: pH increased the most in C+ cultures, in the same time the highest HA concentration was measured in this culture. It should be explained in a more detailed way.
Wu, M., Wu, G., Lu, F. et al. Microalgal photoautotrophic growth induces pH decrease in the aquatic environment by acidic metabolites secretion. Biotechnol Biofuels 15, 115 (2022). https://doi.org/10.1186/s13068-022-02212-z).
The possible role of microalgae in the repression of antibiotic resistant gene abundances should be explained in a little bit more detailed way.
Author Response
Porto, 13th February 2023
Dear Reviewer 1 of Applied Sciences,
Thank you for the opportunity to resubmit a new version of the manuscript applsci-2210602 entitled “Microalgal systems, a green solution for wastewater conventional pollutants removal, disinfection, and reduction of antibiotic resistance genes prevalence?”. Attached, please find a revised version of the manuscript, in which the comments and suggestions were considered to the furthest extent possible. The changes made relative to the resubmitted manuscript are indicated as track changes in the revised version of the document and transcribed below whenever possible.
P1L15-16 (and later throughout the manuscript): In my opinion, the principle to consider antibiotic resistance genes as pollutants is not correct. It suggest that these genes are present freely in wastewaters and cause similar problems as other organic pollutants, but the situation is far from this. I also don't think it is right to call coliform bacteria as pollutants. I think I understand why Authors use these terms, but some scientifically more relevant terms should be find to describe these agents.
Answer: The authors agree with the reviewer’s suggestion, and the designation of coliform bacteria and ARGs as pollutants was revised in the manuscript.
P1L17-18 (and later throughout the manuscript): I do not agree, that the disappearance of coliform and other bacteria carrying antibiotic resistance genes because of the presence of C. vulgaris or other algae could be called “removal”, unless these algae feed on these bacteria, which is not characteristic to green algae, Chlorella among them. Even in the case of toxic or allelopathic compounds, the reduction of the proliferation of these bacteria could be the more correct term (as it is mentioned
Answer: The authors agree with the reviewer, and the term “removal” was corrected to “reduction of the proliferation” in the manuscript.
Beside this, I feel Materials and Methods not detailed enough, and the discussions and explanations of some results are poor. My detailed comments are the following:
Introduction
P1L31: “… natural water bodies.”
Answer: This designation was corrected in the manuscript.
P2L45: The literature cited in the text should be numbered consecutively. So it is not correct that citation 13 is followed by citation 48.
Answer: This citation enumeration was corrected accordingly.
P2L55-56: The sentence is a little bit misleading: it sounds as microalgae would generally produce humic substances. If this is the situation, there should be some more related example from the literature (e.g. from the cited ones, if they present such examples).
Answer: Algal EPS are primarily composed of polysaccharides, proteins, lipids, and nucleic acids but also have humic acid-like substances that may be involved in redox processes (Jiao et al., 2022). Besides, some microalgal species naturally secrete humic acids, while others secret humic acid-like substances along with EPS release in the presence of certain stressors (Jiao et al., 2022). This information was added to Section 3.3 of the manuscript to better understand how the presence of humic substances can be related to microalgae and the reduction of proliferation of some bacteria.
Materials and Methods
Nitrogen is as important for microbial growth as phosphorous. What was the reason not to measure nitrogen forms, at least inorganic ones, assuming that algae are using meanly them as nitrogen sources?
Answer: The Brucine method was conducted to determine the concentration of nitrate-nitrogen (NO3-N) throughout the experiment. However, values were below the detection limit (0.2 mg L-1). For that reason, these results were not presented in the manuscript. However, C, N, and P microalgal assimilation mechanisms are linked and affect each other, mostly because they are the macronutrients composing the typical molar ratios of microalgae (C106H181O45N16P) (Su, 2021).
Hence, due to the straight relationship between N and P assimilation by microalgae, authors believe that presenting the removal of PO4-P is enough to prove the ability of C. vulgaris and AMS species to perform wastewater bioremediation.
P3L102: Why continuous irradiation was used for the algal experiments? It does not represent “real life” situation.
Answer: It may not represent a real-life scenario if it is considered that the only light source is the sun. However, it has been proven that microalgal N and P uptake efficiencies are higher when a continuous light supply is applied, and the absence of a dark period reduces the proliferation of bacteria (Su, 2021). Nonetheless, authors predict the implementation of microalgal systems operating in closed photobioreactors at which light is provided by an artificial source such as LEDs (with high energy efficiency, as with LED, it is possible to provide the optimum light spectrum for microalgal growth).
P3L103-113: There was two C. vulgaris cultures with UWW. Why just two? There was one positive control (with sterile UWW) and one negative control (with original UWW). There should be one without the algae and with sterile UWW. What was the reason not to use such control?
Answer: Biological duplicates were used, from which each sampling was performed in duplicate, performing a n=4. Authors understand that using a control without the algae and with sterile UWW would reveal if other COD and phosphorus removal mechanism would occour, for eg. by photocatalysis. The authors believe that in the absence of any microalgae and bacteria , the effects of other removal mechanisms would be negligible when compared to microalgae and bacteria effects on COD and phosphorus’ removal rate. Moreover, it would be a control of the positive control, not adding any extra valuable information in the scope of this study.
To avoid dilution effect, filtered (or centrifuged) Chlorella medium should be added to controls in the same volume.
Answer: The authors agree and thank the reviewer for the suggestion. Indeed, in the current experiments, this procedure has been followed. However, in the submitted manuscript work, it was not performed and could not be neglected along with the discussion of the results to obtain valid conclusions. Nevertheless, the authors believe that it did not affect the main work conclusions.
2.3 and 2.4 subchapters:
The volumes of collected samples are not indicated.
Answer: Information about the sampling volume was added.
Were the missing volumes removed during sampling refilled to the cultures in some form, or not? If not, could the decreasing volume affect the results? Please, explain.
Answer: The volumes were not refiled; only evaporation effects were restored daily by adding deionized sterilized water. The total sampled volume was less than 10%, which is preconized as the limit to obtain valid results. Furthermore, the volume decrease was the same for the control PBRs.
2.9 subchapter:
How many repetitions were carried out? The mentioned analyses of variances can be carried out only with at least three repetitions.
Answer: The sampling of each PBRs (including individual biological replicas) was performed in duplicate (n=2). Additionally, analytical procedures were also performed in duplicate, performing a total of at least n=4.
Results and Discussion
P7L240-243: In my opinion COD is not removed, but decreased (because chemical oxygen demand cannot be accumulated or adsorbed as phosphate).
Answer: The authors agreed with the reviewer and modifications were performed to the manuscript.
P8L278-283: It is hard to understand, how humic acid concentration connected to the presence of Chlorella vulgaris. Is there any evidence that green algae are secreting humic substances (or contribute to the presence of humic substances) in significant extent? In a recent study, C. vulgaris was present as a control (the one which less related to humic substances; Wu et al., 2022).
Answer: The authors understand the reviewer’s question. EPS content in wastewater may have several sources. However, as reported by Chen et al., 2015, algae may also have a role in the production of EPS, from which humic acids are a component “ EPS is a kind of the macromolecular substance generated by organisms (such as algae), mainly composed of proteins, polysaccharides, humic substances, lipids, nucleic acid, etc., originating from microbial metabolism, cell lysis, hydrolysis of macromolecules and sorption of constituents from wastewater (Chen et al., 2015, Sheng et al., 2010)”.
Additionally, there are several pieces of evidence related to EPS production from Chlorella sp., including studies applied for wastewater treatment (Chen et al., 2015, Zhang et al., 2021, Barboríková et al., 2019). Furthermore, according to Wu et al., 2022, although Chlorella vulgaris (CV) was used as a control group, they also reported that CV could produce DOM with content in humic acids, although it was produced to a much lesser extent than Euglena sp. as transcribed below:
“What is more, the DOM content of EG (29.9 mg cell−1 × 10–6) was 49.8-fold that of CV (0.6 mg cell−1 × 10–6) (Fig. 2C, quantification by single cells, p < 0.001), indicating that a large amount of DOM was secreted from EG cells. The analysis of 3D-EEM spectroscopy showed that these DOM were mainly composed of humic acid, and EG had a significantly higher fluorescence intensity than that of CV (Fig. 2D, E), indicating that EG secreted a large number of humic acids than that of CV” (Wu et al., 2022).
In our study, it was observed the same HA,COD and microalgae growth profile over time, with high correlation between HA and MCC in both CV and C- PBRS. Nonetheless, a correlation was found between HA and COD, but in a lesser extent (r2 0.7-0.8). Hence, these results led us to conclude that although a few studies directly not report the HA content with C. vulgaris, it can be correlated.
Furthermore: pH increased the most in C+ cultures, in the same time the highest HA concentration was measured in this culture. It should be explained in a more detailed way.
Answer: We understand this observation, since Wu et al. (2022) reported an accentuated decrease in pH when Euglena sp. excreted more HA. However, it should be noted that the production of several other acid compounds by Euglena sp. was also reported, such as itaconic acid, that all together contributed to the decrease of pH.
In our study, in CV PBRs, although a peak of HA production has been observed, it does not mean that the attained HA concentration was enough to decrease the pH. Furthermore, other cellular events can contribute to the pH increase, such as the consumption of HCO3- by algae (as a result of photosynthesis), ” the photosynthetic process of microalgae induces pH increase by protons (H+) uptake independently in the aquatic environment”(Wu et al., 2022). Wu et al. (2022) also pointed out: “we put forward a new point of view: the increase in the pH of the photosynthetic autotrophic aquatic environment of microalgae was due to not only the high photosynthetic efficiency of microalgae and the absorption of CO2 in the aquatic environment but also the microalgae cells not secreting a lot of acidic metabolites.” Additionally, it should be noted that a high correlation was found in C+ between MCC and pH, indicating that microalgal growth had a main role in the pH variation.
The possible role of microalgae in the repression of antibiotic resistant gene abundances should be explained in a little bit more detailed way
Answer: The reduction of intI1 and ARG can occur via: (i) the reduction of proliferation of antibiotic-resistant bacteria and bacteriophages carrying the ARG, or (ii) the loss of ARG-containing plasmids [40,42].
Also, recently, Tang et al. (2022) observed that a C. vulgaris -B. licheniformis consortium was able to promote sul1 reduction of prevalence due to the efficient removal of exogenous ARGs plasmids carrying sul1. The authors observed that microalgal cells: (i) absorbed and endocytosed the exogenous ARGs plasmids carrying sul1, with no affection of the microalgae cell nucleus; (ii) EPS promoted and increased the adsorption of exogenous ARGs plasmids; (iii) intracellular accumulation of ARGs may have induced the cells to produce increasing contents of cross-linking agents disrupting the exogenous DNA replication; and (iv) endocytosis increased with a larger time (more than 6 days) of contact between microalgae and plasmids.
However, in our study, these events were not evaluated; hence conclusions regarding the microalgal mechanisms towards the reduction of ARGs prevalence are highly speculative. Furthermore, the study of Tang et al. (2022) was performed in synthetic media instead of real wastewater, with a controlled microbial population and different operating conditions (temperature, light exposure, and retention time). However, in a real scenario using real wastewater, there are no guarantees that the same mechanisms may occur due to the complexity of wastewater composition and the intracellular presence of plasmids. Hence, the authors prefer not to include this information as a possible explanation and consider the co-precipitation of ARB with microalgae as a more reasonable explanation so far. However, due to the promising results of AMS and the lack of information on microalgal mechanisms responsible for ARGs reduction, this will be considered in a further study.
References
BARBORÍKOVÁ, J., ŠUTOVSKÁ, M., KAZIMIEROVÁ, I., JOŠKOVÁ, M., FRAŇOVÁ, S., KOPECKÝ, J. & CAPEK, P. 2019. Extracellular polysaccharide produced by Chlorella vulgaris – Chemical characterization and anti-asthmatic profile. International Journal of Biological Macromolecules, 135, 1-11.
CHEN, B., LI, F., LIU, N., GE, F., XIAO, H. & YANG, Y. 2015. Role of extracellular polymeric substances from Chlorella vulgaris in the removal of ammonium and orthophosphate under the stress of cadmium. Bioresource Technology, 190, 299-306.
JIAO, Y., ZHU, Y., CHEN, M., WAN, L., ZHAO, Y., GAO, J., LIAO, M. & TIAN, X. 2022. The humic acid-like substances released from Microcystis aeruginosa contribute to defending against smaller-sized microplastics. Chemosphere, 303, 135034.
SHENG, G.-P., YU, H.-Q. & LI, X.-Y. 2010. Extracellular polymeric substances (EPS) of microbial aggregates in biological wastewater treatment systems: A review. Biotechnology Advances, 28, 882-894.
SU, Y. 2021. Revisiting carbon, nitrogen, and phosphorus metabolisms in microalgae for wastewater treatment. Science of The Total Environment, 762, 144590.
TANG, Y., SONG, L., JI, X., HUANG, S., YU, Y., YE, J., XU, W. & HOU, M. 2022. Algal-bacterial consortium mediated system offers effective removal of nitrogen nutrients and antibiotic resistance genes. Bioresource Technology, 362, 127874.
WU, M., WU, G., LU, F., WANG, H., LEI, A. & WANG, J. 2022. Microalgal photoautotrophic growth induces pH decrease in the aquatic environment by acidic metabolites secretion. Biotechnology for Biofuels and Bioproducts, 15, 115.
ZHANG, Y., LI, X., DU, J., PU, L. & CHEN, S. 2021. Effects of Chlorella extracellular polymeric substances on the aggregation and stability of TiO2 nanoparticles as electrolytes. Desalination and Water Treatment, 209 334–341.
Author Response File: 
Author Response.docx
Reviewer 2 Report
In the manuscript entitled "Microalgae systems, a green solution for wastewater conventional pollutants removal, disinfection, and reduction of antibiotic resistance genes prevalence?" the authors evaluated the ability of Chlorella vulgaris and UWW autochthonous microalgal species to simultaneously remove PO4--P, coliforms, and ARGs. Overall, this manuscript is demonstrating an important and promising research direction, however part of this manuscript seems to be enhanced in much clearer discussion. Herein, it is suggested that the manuscript could be accepted for publication in Applied Sciences unless major corrections has been conducted according to the recommended points.
Required corrections:
1) Key words: microalgae should be changed into microalgae technology.
2) Introduction section:
Line 49, namely with microalgae should be changed into such as microalgae technology.
3) Besides, the current research and problems should also be summarized in this section, especially in the third paragraph.
4) In the Results and discussion section: it is suggested that the authors should list the advantages and comparison of this study through comparing the current studies, and explain the reason.
5) In addition, there are two related papers you need to include in the Discussion sections.
(a) Bioengineering, 2022, 9(11), 637;
(b) Journal of Cleaner Production, 2022, 355, 131768.
6) The future perspective should be summarized in this manuscript.
7) There are many format errors which suggested the authors need to be modified carefully throughout the whole manuscript.
8) The English language should be revised by an expert.
Author Response
Porto, 13th February 2023
Dear Reviewer 2,
Thank you for the opportunity to resubmit a new version of the manuscript applsci-2210602 entitled “Microalgal systems, a green solution for wastewater conventional pollutants removal, disinfection, and reduction of antibiotic resistance genes prevalence?”. Attached please find a revised version of the manuscript, in which the comments and suggestions were considered to the furthest extent possible. The changes made relative to the resubmitted manuscript are indicated as track changes in the revised version of the document and transcribed below whenever possible.
Required corrections:
1) Keywords: microalgae should be changed into microalgae technology.
Answer: The reviewer's suggestion was performed accordingly.
2) Introduction section:
Line 49, namely with microalgae should be changed into such as microalgae technology.
Answer: The reviewer's suggestion was performed accordingly.
3) Besides, the current research and problems should also be summarized in this section, especially in the third paragraph.
Answer: The authors understand the importance of such information. However, we believe that it is already included in other paragraphs. In the first paragraph, the main problems are described as being related to the ineffectiveness of the current methods implemented in UWWTP, the high energy demand of currently applied tertiary treatments, and the need for adding chemicals, which may imply the generation of other harmful by-products. Furthermore, current research about the uses of microalgae as a tertiary treatment is summarized in the 3rd paragraph concerning the potential to reduce pathogens proliferation and ARGs abundance. Nonetheless, the following information was added:
Lines 50-54:- “Biological tertiary treatments, namely with microalgae technology, appear as a more environmentally friendly alternative, particularly due to their high metabolic flexibility and the non-generation of toxic by-products. Additionally, the biomass obtained at the end of the process can be converted to commercially valued products, such as bio-fuels or biofertilizers, paying off the tertiary treatment implementation’s costs. “
Lines 58-60: “ Particularly, light exposure above 115 μmol m−2 s−1 has a crucial role in E. coli removal, in combination with DO values between 1.2-8.18 mg L-1 and alkaline pH (above 8.5) [19,20].”
4) In the Results and discussion section: it is suggested that the authors should list the advantages and comparison of this study through comparing the current studies, and explain the reason.
(a) Bioengineering, 2022, 9(11), 637;
(b) Journal of Cleaner Production, 2022, 355, 131768.
Answer: In the authors’ opinion, Gang Li et al. papers are very interesting. However, “Cultivation of microalgae in adjusted wastewater to enhance biofuel production and reduce environmental impact: Pyrolysis performances and life cycle assessment” and “Enhancing Bioenergy Production from the Raw and Defatted Microalgal Biomass Using Wastewater as the Cultivation Medium” are not in the scope of the submitted manuscript.
6) The future perspective should be summarized in this manuscript.
Answer: The authors followed the reviewer’s suggestion, and the following information was added in the conclusion section:
“For future work, it should be interesting to identify microalgal and bacterial species in the AMS system. Also, monitoring the AMS species and wastewater composition, namely in terms of nitrogen and phosphorus, would be helpful in understanding the dynamics of this system. As discussed throughout the present work, abiotic factors such as light intensity have a main role in both microalgal growth and the reduction of pathogens’ proliferation, namely in conjugation with pH. Hence, studies considering the light intensity and pH ranges would be helpful in optimizing the potential of AMS. Moreover, due to the scarcity of information related to the microalgal mechanisms involved in ARGs reduction of prevalence in real wastewater, it should be promising to evaluate these events in future studies.”
7) There are many format errors which suggested the authors need to be modified carefully throughout the whole manuscript. 8) The English language should be revised by an expert.
Answer: English language and format errors were revised in the manuscript.
Author Response File: Author Response.docx
Round 2
Reviewer 1 Report
During the revision, the Authors answered most of the questions that arose. Nonetheless, I can not agree with some answers – e.g. that measuring only phosphorous could give enough information about removal capacities of an alga, or that using photobioreactors for tertiary wastewater treatment would be the most sustainable way in the future. My main problem is not this (I understand that some of them can not be changed – e.g. measurement of nitrogen forms). The main problem is that the broader explanations of these issues were not involved in the revised manuscript. The authors acceptably explained some of the issues as an answer to the reviewer, but the revision of the manuscript itself did not happened (the Materials and Methods section and the Results and Discussion section remained almost the same as in the previous version. So I can not consider the author’s work as “major revision”. Again: I can accept the answers, but at least part of them should be involved into the manuscript, otherwise the questions arose in the first round remain unanswered.
Author Response
Dear Reviewer 1,
Thank you for the opportunity to resubmit a new version of the manuscript applsci-2210602 entitled “Microalgal systems, a green solution for wastewater conventional pollutants removal, disinfection, and reduction of antibiotic resistance genes prevalence?”. Attached, please find a revised version of the said manuscript, in which the comments and suggestions were considered to the furthest extent possible. The changes made relative to the resubmitted manuscript are indicated as track changes in the revised version of the document and transcribed below whenever possible.
Required corrections:
During the revision, the Authors answered most of the questions that arose. Nonetheless, I can not agree with some answers – e.g. that measuring only phosphorous could give enough information about removal capacities of an alga, or that using photobioreactors for tertiary wastewater treatment would be the most sustainable way in the future. My main problem is not this (I understand that some of them can not be changed – e.g. measurement of nitrogen forms). The main problem is that the broader explanations of these issues were not involved in the revised manuscript. The authors acceptably explained some of the issues as an answer to the reviewer, but the revision of the manuscript itself did not happened (the Materials and Methods section and the Results and Discussion section remained almost the same as in the previous version. So I can not consider the author’s work as “major revision”. Again: I can accept the answers, but at least part of them should be involved into the manuscript, otherwise the questions arose in the first round remain unanswered.
Answer:
-The author agrees with the reviewer’s suggestions; hence information related to nitrogen content and possible assimilation was added in the Materials and Methods and Discussion sections as follows:
Line 99: “(iv) nitrogen concentration based on nitrate-nitrogen levels (NO3-N), according to the United States Environmental Protection Agency (USEPA)-approved Brucine Colorimetric Method [1].”
|
Table 1: Nitrate–nitrogen (NO3–N) |
< LOD 1 |
mg L−1 |
Line 150: “Due to the non-detected concentration of NO3–N (< LOQ ) in the raw wastewater it was not quantified during the experiment.”
Line 258: “Microalgae molar ratios are typically composed of C, N, and P (C106H181O45N16P), and their assimilation mechanisms are linked [2].”
Line 308: “Although it was not possible to calculate NO3-N removal efficiencies, due to the linkage between the macronutrients, it is expected that either C. vulgaris and AMS could remove this pollutant as well as other species of nitrogen such as NH4 [3-5].”
-Concerning the reviewer’s comment, “photobioreactors for tertiary wastewater treatment would be the most sustainable way in the future”, the authors understand that the use of open retention ponds or raceways is the most economic solution to implement tertiary microalgal treatment. However, the aim of this study did not focus on the use of PBRs; it only used closed PBRs to most properly control all the variables, particularly concerning the microbiological ones. This study is still preliminary, and a wide variety of variables need to be optimized before implementing a microalgal tertiary treatment. Although, the authors mentioned in the former letter that the use of artificial light was considered to predict an “implementation of microalgal systems operating in closed photobioreactors”, this is not included in the aim of the study, is just a predicted step forward towards higher energy efficiency. Nevertheless, the use of artificial light can also be implemented in open raceways [6].
- Information related to the evaporation effects was added in line 126 ” Evaporation effects were restored daily by adding deionized sterilized water.”
- More clear information about humic acids and microalgae was added in line 332: “Some microalgal species naturally secrete humic acids, while others secret humic acid-like substances along with EPS release in the presence of certain stressors [7,8].”
REFERENCES
- EPA, U. Method 352.1: Nitrogen, nitrate (colorimetric, brucine) by spectrophotometer; DC, USA, 1971.
- Su, Y. Revisiting carbon, nitrogen, and phosphorus metabolisms in microalgae for wastewater treatment. Science of The Total Environment 2021, 762, 144590, doi:https://doi.org/10.1016/j.scitotenv.2020.144590.
- Salgado, E.M.; Gonçalves, A.L.; Sánchez-Soberón, F.; Ratola, N.; Pires, J.C.M. Microalgal cultures for the bioremediation of urban wastewaters in the presence of siloxanes. International Journal of Environmental Research and Public Health 2022, 19, 2634, https://www.mdpi.com/1660-4601/19/5/2634.
- Yu, H.; Kim, J.; Lee, C. Nutrient removal and microalgal biomass production from different anaerobic digestion effluents with chlorella species. Scientific Reports 2019, 9, 6123, doi:10.1038/s41598-019-42521-2.
- Whitton, R.; Le Mével, A.; Pidou, M.; Ometto, F.; Villa, R.; Jefferson, B. Influence of microalgal n and p composition on wastewater nutrient remediation. Water Research 2016, 91, 371-378, doi:https://doi.org/10.1016/j.watres.2015.12.054.
- Carneiro, M.; Maia, I.B.; Cunha, P.; Guerra, I.; Magina, T.; Santos, T.; Schulze, P.S.C.; Pereira, H.; Malcata, F.X.; Navalho, J.; et al. Effects of led lighting on nannochloropsis oceanica grown in outdoor raceway ponds. Algal Research 2022, 64, 102685, doi:https://doi.org/10.1016/j.algal.2022.102685.
- Wu, M.; Wu, G.; Lu, F.; Wang, H.; Lei, A.; Wang, J. Microalgal photoautotrophic growth induces ph decrease in the aquatic environment by acidic metabolites secretion. Biotechnology for Biofuels and Bioproducts 2022, 15, 115, doi:10.1186/s13068-022-02212-z.
- Jiao, Y.; Zhu, Y.; Chen, M.; Wan, L.; Zhao, Y.; Gao, J.; Liao, M.; Tian, X. The humic acid-like substances released from microcystis aeruginosa contribute to defending against smaller-sized microplastics. Chemosphere 2022, 303, 135034, doi:https://doi.org/10.1016/j.chemosphere.2022.135034.
Author Response File: Author Response.docx
Reviewer 2 Report
The revised manuscript is clear and significantly improved. Acceptance is recommended unless some modifications that listed as below:
(1) The introduction should be demonstrated the current research about algae with wastewater treatment, especially both the advantages and disadvantages, and with the nowadays applications, such as bioenergy production by microalgae with wastewaster treatment. It is suggested that some recent literatures could be referred to, such as (a) and (b): (a) Bioengineering, 2022, 9(11), 637;
Author Response
Dear Reviewer 2,
Thank you for the opportunity to resubmit a new version of the manuscript applsci-2210602 entitled “Microalgal systems, a green solution for wastewater conventional pollutants removal, disinfection, and reduction of antibiotic resistance genes prevalence?”. Attached, please find a revised version of the said manuscript, in which the comments and suggestions were considered to the furthest extent possible. The changes made relative to the resubmitted manuscript are indicated as track changes in the revised version of the document and transcribed below whenever possible.
Required corrections:
1) The introduction should be demonstrated the current research about algae with wastewater treatment, especially both the advantages and disadvantages, and with the nowadays applications, such as bioenergy production by microalgae with wastewaster treatment. It is suggested that some recent literatures could be referred to, such as (a) and (b): (a) Bioengineering, 2022, 9(11), 637;
Answer: As suggested by the reviewer, the following papers were added to the manuscript:
Gang Li et al. “Enhancing Bioenergy Production from the Raw and Defatted Microalgal Biomass Using Wastewater as the Cultivation Medium”;
Álvarez-González et al. “Can microalgae grown in wastewater reduce the use of inorganic fertilizers?”
Author Response File: Author Response.docx
