Growth Conditions and Growth Kinetics of Chlorella Vulgaris Cultured in Domestic Sewage
Round 1
Reviewer 1 Report
The author of the manuscript sustainability-2003216 intended to analyze the growth pattern of Chlorella in wastewater. This topic is actual – in Web of Science Clarivate Analytics, 263 papers related to wastewater and Chlorella were published this year till now.
The manuscript has flaws in design, methodology, interpretation, and writing. It is intended to be a Study protocol. A Study Protocol is a detailed research for research projects that just started or will be started. The Study Protocol must not include results – because the results were not yet generated. According to the MDPI announcement of Study Protocol type of article, “Authors are encouraged to later publish the study results as research articles, in any journal of MDPI…. The research article and the original study protocol will then be linked.” The author presents results and not only a detailed plan of research.
The experiment design is intended to analyze “three types of growth conditions, initial pH, light intensity, and algae addition” in “term of growth kinetic.” The analysis is done without replicates and/or statistical tools.
The author uses the illuminance unit – lux. He must use the unit of Photosynthetically Active Radiation (PAR), i.e., µmoles of photons (Einsteins) per square meter per second (µE m-2 s-1)., across the spectral range of 400 to 700 nm. The author does not provide information regarding the light source.
The experiment is not adequately presented – the author made all the reported experiments with the same wastewater processed by filtration “in a 1.2mm GF/C filter unit to prevent the suspended material in the sewage samples from affecting the growth of Chlorella vulgaris”? What means “photobioreactor built in the medium”? The Chlorella vulgaris culture was xenic or axenic? The author does not detail in the Material and Methods Section the methods used to analyze nutrients in wastewater.
Modeling is done on data that do not have statistical analysis.
Writing is not rigorous. Reference are missing – i.e., L168, [Error! Reference source not found.]
Scientific names must be written in Italics.
Author Response
The author of the manuscript sustainability-2003216 intended to analyze the growth pattern of Chlorella in wastewater. This topic is actual – in Web of Science Clarivate Analytics, 263 papers related to wastewater and Chlorella were published this year till now.
1. The manuscript has flaws in design, methodology, interpretation, and writing. It is intended to be a Study protocol. A Study Protocol is a detailed research for research projects that just started or will be started. The Study Protocol must not include results – because the results were not yet generated. According to the MDPI announcement of Study Protocol type of article, “Authors are encouraged to later publish the study results as research articles, in any journal of MDPI…. The research article and the original study protocol will then be linked.” The author presents results and not only a detailed plan of research.
Response: This paper not only presents a detailed research plan, but also describes the complete research process, and based on the results, proposes suggestions for developing more sophisticated growth kinetic models.
2. The experiment design is intended to analyze “three types of growth conditions, initial pH, light intensity, and algae addition” in “term of growth kinetic.” The analysis is done without replicates and/or statistical tools.
Response: Use spss software to process the analysis data and carry out the significance test of the parameters (Table 3 significance test results). Then use the original software for image plotting. In addition, the study solves the growth model using spss nonlinear regression techniques combined with image plots to assess the confidence level of the model.
3. The author uses the illuminance unit – lux. He must use the unit of Photosynthetically Active Radiation (PAR), i.e., µmoles of photons (Einsteins) per square meter per second (µE m-2 s-1)., across the spectral range of 400 to 700 nm. The author does not provide information regarding the light source.
Response: The light source is an LED lamp with a spectral range of 400-700nm. Light intensity units have been modified to μF N-2 s-1
4. The experiment is not adequately presented – the author made all the reported experiments with the same wastewater processed by filtration “in a 1.2mm GF/C filter unit to prevent the suspended material in the sewage samples from affecting the growth of Chlorella vulgaris”? What means “photobioreactor built in the medium”? The Chlorella vulgaris culture was xenic or axenic? The author does not detail in the Material and Methods Section the methods used to analyze nutrients in wastewater.
response:
(1) In order to prevent the effect of suspended solids in domestic wastewater on the growth of Chlorella, the wastewater samples were first allowed to stand for 24 hours, and then the supernatant of the wastewater samples was filtered with a GF/C filter with a pore size of 1.2 mm.
(2) The experimental procedure was carried out in an in-house built photobioreactor. See Figure 1.
(3) Chlorella was kept in BG11 for cultivation, and the cultivation process was carried out in a constant temperature light incubator with an illumination intensity of 4000 lux, an illumination time of 24 h, and a temperature of 25 °C. It belongs to the autotrophic-heterotrophic mixed culture mode.
(4) The analytical method of nutrients in wastewater has been described in detail in 2.2.
5. Modeling is done on data that do not have statistical analysis.
Response: SPSS software one-way analysis of variance significance test was performed on the data entered into the three types of growth kinetic models (Table 3 significance test results).
6. Writing is not rigorous. Reference are missing – i.e., L168, [Error! Reference source not found.]
Response: The question where the reference has been cited has been re-corrected.
7. Scientific names must be written in Italics.
Response: Modified.
Author Response File: 
Author Response.docx
Reviewer 2 Report
The authors introduced growth kinetic models to analyze the growth pattern of Chlorella in domestic wastewater. The work is important for achieving sustainable development goals. Several comments need to be considered before publishing this work.
1. Why only pH values, light intensity, and algae addition factors were considered in this study? What about different element contents of wastewater from various sources?
2. For the statistical analysis, did the authors study the statistically significant differences among different factors? why this is not mentioned in the figures?
3. Several articles were published on this topic, what is the novelty of this work, authors should add that at the end of the introduction section.
Similar published articles
- Lavrinovičs, A., Mežule, L., Cacivkins, P., & Juhna, T. (2022). Optimizing phosphorus removal for municipal wastewater post-treatment with Chlorella vulgaris. Journal of Environmental Management, 324, 116313.
- Manhaeghe, D., Michels, S., Rousseau, D. P., & Van Hulle, S. W. (2019). A semi-mechanistic model describing the influence of light and temperature on the respiration and photosynthetic growth of Chlorella vulgaris. Bioresource technology, 274, 361-370.
- Lam, M. K., Yusoff, M. I., Uemura, Y., Lim, J. W., Khoo, C. G., Lee, K. T., & Ong, H. C. (2017). Cultivation of Chlorella vulgaris using nutrients source from domestic wastewater for biodiesel production: Growth condition and kinetic studies. Renewable Energy, 103, 197-207.
Author Response
1.Why only pH values, light intensity, and algae addition factors were considered in this study? What about different element contents of wastewater from various sources?
Response:
(1) pH, light intensity, and algae addition had significant effects on the growth of Chlorella vulgaris.
(2) The study selected secondary treated water from the wastewater plant as the medium to assess the feasibility of achieving the dual objectives of domestic wastewater treatment and biomass accumulation. pH, light intensity, and algae addition are all factors that can be easily controlled in the treatment technology. The remaining influencing factors such as temperature were relatively difficult to control and therefore were not considered.
2.For the statistical analysis, did the authors study the statistically significant differences among different factors? why this is not mentioned in the figures?
Response: Sorry, the significance analysis results for each factor were not initially recorded in the article due to space considerations. They are now added after 3. Results and Discussion (Table 3 Significance Test Results).
3. Several articles were published on this topic, what is the novelty of this work, authors should add that at the end of the introduction section.
Response: The innovation and research significance of the article are added at the end of the introduction.
Reviewer 3 Report
Before acceptance the paper needs improvements.
1. Introduction is very short . Please complete it.
2. Figures 2, 3,7 an 8 not mentioned in text. Please do it. Also renumber them.
3. Strange numbering of tables. Please renumber them.
4. Date mean days in fig. 2,4 and 6? If yes, change label.
5. How this process can be implemented?
Author Response
- Introduction is very short . Please complete it.
Reply: Supplements have been made. - Figures 2, 3,7 an 8 not mentioned in text. Please do it. Also renumber them.
Response: Modified. - Strange numbering of tables. Please renumber them.
Answer: It has been revised and renumbered. - Date mean days in fig. 2,4 and 6? If yes, change label.
Response: The label has been modified. - How this process can be implemented?
Response: If this process refers to the entire experimental process, then the "2. Materials and methods" section details the methods and procedures used to conduct the study.
Reviewer 4 Report
Line 9 This is an incomplete sentence
Line 12 Identify the other models
Line 18 and throughout the paper type out the words nitrogen, phosphorus, and carbon
Line 34 treatment; it can
Line 39 environment; therefore, three
Line 44 used kinetic growth models
Line 50 Chlorella culture?
Line 60 point. The water
Line 64 Define TN, TP and COD
Line 67 photobioreactor, shown in Fig. 1, which
Line 76 Chlorella reached
Line 77 was stopped
Line 77 Chlorella settled
Line 77 layers formed; the upper layer contained water with suspended
Line 79 contained concentrated
Line 96 and biomass production
Line 97 (2) and (3)
Line 100 This is an incomplete sentence
Line 102 different volumes of
Line 112 Define the term variable Ѵ
Line 126 calc
Line 170 The reference is missing
Line 176 trend, and algal
Line 194 depletion; this will be
Line 201 in the 0.20 v/v?
Figure 7 Should temperature be replaced with Density of microalgae?
Line 215 Remove situation
Line 223 respectively; the removal
Line 242 (e)
Table 5 0.10 v and 0.20v?
Line 256 increased. Although
Discuss controls
Author Response
Dear Reviewers:
We are very grateful to Reviewer for reviewing the paper so carefully. And we have carefully considered the suggestion of Reviewer and make some changes.
1、Line 9 This is an incomplete sentence
Response：Modified
2、Line 12 Identify the other models
3、Line 18 and throughout the paper type out the words nitrogen, phosphorus, and carbon
Response：Modified
4、Line 34 treatment; it can、
Response：Modified
5、Line 39 environment; therefore, three
Response：Modified
6、Line 44 used kinetic growth models
Response：Modified
7、Line 50 Chlorella culture?
Response：“Chlorella vulgaris was preserved and cultivated in BG11”
8、Line 60 point. The water
Response：Modified
9、Line 64 Define TN, TP and COD
Response：The TN (Total Nitrogen), TP (Total phosphorus), NH3-N and COD (Chemical oxygen demand)
10、Line 67 photobioreactor, shown in Fig. 1, which
Response：Modified
11、Line 76 Chlorella reached
Response：Modified
12、Line 77 was stopped
Response：Modified
13、Line 77 Chlorella settled
Response：Modified
14、Line 77 layers formed; the upper layer contained water with suspended
Response：Modified
15、Line 79 contained concentrated
Response：Modified
16、Line 96 and biomass production
Response：Modified
17、Line 97 (2) and (3)
Response：Modified
18、Line 100 This is an incomplete sentence
Response：Modified
19、Line 102 different volumes of
Response：Modified
20、Line 112 Define the term variable Ѵ
Response：Modified
21、Line 126 calc
Response：Modified
22、Line 170 The reference is missing
Response：References checked
23、Line 176 trend, and algal
Response：Modified
24、Line 194 depletion; this will be
Response：Modified
25、Line 201 in the 0.20 v/v?
Response：“in the 0.02v/v experimental group”
26、Figure 7 Should temperature be replaced with Density of microalgae?
Response：Modified
27、Line 215 Remove situation
Response：Modified
28、Line 223 respectively; the removal
Response：Modified
29、Line 242 (e)
响应：已修改
30、表5 0.10V和0.20V？
Response：“0.10v/v...”
31. Line 256 increased
Discuss controls
Response：Modified
Round 2
Reviewer 1 Report
he author of the manuscript sustainability-2003216 made efforts to improve it. However, the manuscript still has design, methodology, and writing flaws. Authors claimed the introduction of the statistics – ” Use spss software to process the analysis data and carry out the significance test of the parameters (Table 3 significance test results). Then use the original software for image plotting. In addition, the study solves the growth model using spss nonlinear regression techniques combined with image plots to assess the confidence level of the model”. However, the statistics method is not described. The number of replicates is not presented. The graphics are not considering statistical errors.
The variability of the wastewater is not determined – both in terms of chemical and microbiological composition. The influence of the bacteria from the phycosphere in the mixotrophic growth of the microalgae is not considered – despite the significant importance of the phycopshere bacteria and their interaction with bacteria from wastewater. Jiang, L., Li, Y., & Pei, H. (2021). Algal–bacterial consortia for bioproduct generation and wastewater treatment. Renewable and Sustainable Energy Reviews, 149, 111395. Sial, A., Zhang, B., Zhang, A., Liu, K., Imtiaz, S. A., & Yashir, N. (2021). Microalgal–bacterial synergistic interactions and their potential influence in wastewater treatment: A review. BioEnergy Research, 14(3), 723-738.
I reflected for several days on this manuscript, trying to understand and find the reason for publishing it. My opinion is, that in the present form, the manuscript is still unsuitable for publication.
Author Response
Dear Reviewers:
We are very grateful to Reviewer for reviewing the paper so carefully. And we have carefully considered the suggestion of Reviewer and make some changes.
Reviewer 3 Report
The paper can be accepted for publication.
Author Response
Dear Reviewers:
We are very grateful to Reviewer for reviewing the paper so carefully. And we have carefully considered the suggestion of Reviewer and make some changes.
Round 3
Reviewer 1 Report
The manuscript sustainability-2003216 still needs significant improvements before publication.
Cultivation conditions for Chlorella in sewage water are not presented in the Material and methods section. What type of light source was used? How was the intensity measured? The initial culture of Chlorella vulgaris in BG11 was xenic or axenic?
Experiments were not done in replicates - figures 2-8 are without error bars. Despite the lack of replicates, the data were statistically analyzed.
The variability of the used domestic sewage is unusually low. The incertitudes of the analytical methods used for sewage water analysis are not presented.
The microbial burden of the sewage water is not analyzed. The influence of the microbial burden from different batches of sewage water on Chlorella growth is not determined.
The authors did not pay enough attention to details.
- L82 - high-speed centrifuge (5000r/min, 10min) – to assure reproducibility of the experiments by others, the Relative Centrifugal Force (RCF) or g-force must be presented.
- L288 – „light intensity of 12000lux”, although in Lines 143-158, light intensity is presented in μmol·m-2·s-1.
- The scientific name (i.e., Chlorella vulgaris) must be written in Italics. I asked that from my initial review – still not corrected.
Author Response
Dear Reviewers:
We are very grateful to Reviewer for reviewing the paper so carefully. And we have carefully considered the suggestion of Reviewer and make some changes.
1、Cultivation conditions for Chlorella in sewage water are not presented in the Material and methods section. What type of light source was used? How was the intensity measured? The initial culture of Chlorella vulgaris in BG11 was xenic or axenic?
Response : The light source in the constant temperature illumination incubator is LED lamp, and the light intensity in the environment is measured by the light intensity measuring instrument. The initial culture of Chlorella vulgaris in BG11 was axenic.
2、Experiments were not done in replicates - figures 2-8 are without error bars. Despite the lack of replicates, the data were statistically analyzed.
Response: Due to several factors such as algae species purchase and laboratory environment due to seasonal changes, no repeat experiments were set up. Our team believes that the existing experimental data can still support the experimental results.
3、The variability of the used domestic sewage is unusually low. The incertitudes of the analytical methods used for sewage water analysis are not presented.
Response: The study used domestic wastewater taken from the secondary treatment inlet point of the wastewater plant, and the subsequent simple purification operation was designed to reduce the impact on Chlorella culture. The error of the method used for wastewater quality analysis is small, mainly due to human operation, and therefore not mentioned.
4、The microbial burden of the sewage water is not analyzed. The influence of the microbial burden from different batches of sewage water on Chlorella growth is not determined.
Response : This study considers the effects of several easily controlled environmental factors on the growth of Chlorella vulgaris.
5、- L82 - high-speed centrifuge (5000r/min, 10min) – to assure reproducibility of the experiments by others, the Relative Centrifugal Force (RCF) or g-force must be presented.
Response :RCF:1100g
6、- L288 – „light intensity of 12000lux”, although in Lines 143-158, light intensity is presented in μmol·m-2·s-1.
Response: The unit has been unified and revised to 150 μmol·m-2·s-1.
7、- The scientific name (i.e.,Chlorella vulgaris) must be written in Italics. I asked that from my initial review – still not corrected.
Response: Revised in full
