Formulation and Characterization of a Heterotrophic Nitrification-Aerobic Denitrification Synthetic Microbial Community and its Application to Livestock Wastewater Treatment
Round 1
Reviewer 1 Report
In the manuscript, the authors isolated several bacterial strains and used these isolated strains to form a synthetic community. The authors claimed that the synthetic community was able to perform heterotrophic nitrification and aerobic denitrification simultaneously. However, the authors failed to provide data to support either nitrification or denitrification. These isolates grew very fast as the authors showed, and the removal of ammonia could largely due to the assimilation into biomass. As the authors stated in line 379-340, the total nitrogen in the effluent was higher than ammonia nitrogen, this indicated that nitrogen was not removed (at least no denitrification). In addition, as the authors suggested, glucose or succinate was needed as carbon source during the process, this indicated there was limited economic value of applying the synthetic community in wastewater treatment.
Line 2, the title, a mixed culture can not be called as ‘composite strain’, better to use ‘synthetic microbial community’ throughout the manuscript;
Line 15, For wastewater treatment, only one strain was used is hardly true.
All bacterial names need to be in italic
Line38-40, the meaning is unclear, please check the grammar
Line73, change to ‘higher removal efficiency of nitrogen and COD and less nitrous oxide…’
Line 116, what’s the logic of this sentence?
Line 133, the primer is 1492R
Figure 1-4, it is meaningless to show the plate images
Fgiure5, what do the author want to show here? I suggest to substitute the phylogeny with a table indicating to which known bacteria each isolate was closely related, and the identity between their 16S rRNA sequences.
Author Response
Dear Reviewer:
We appreciate your careful review and review of our manuscript. Your comments are significant for the improvement and condensing of our manuscript. Moreover, your comments also point out a lot of interesting research directions, which provides a reference for us to carry out further research in the future. We have studied comments carefully and have made corrections and responses, which we hope to meet with approval. Revised portions are marked in yellow in the paper. The primary corrections in the paper and the responses to the reviewer’s comments are as following:
Comment 1: In the manuscript, the authors isolated several bacterial strains and used these isolated strains to form a synthetic community. The authors claimed that the synthetic community was able to perform heterotrophic nitrification and aerobic denitrification simultaneously. However, the authors failed to provide data to support either nitrification or denitrification.
Response 1: The reason for not providing the data of nitrification and denitrification is that the isolated strains have been identified by DNA identification as strains confirmed to have nitrification and denitrification. Many people have done experiments on this, so they have not been repeated tests. The focus of this experimental research is also the performance study of composite strains.
Comment 2: These isolates grew very fast as the authors showed, and the removal of ammonia could largely due to the assimilation into biomass. As the authors stated in line 379-340, the total nitrogen in the effluent was higher than ammonia nitrogen, this indicated that nitrogen was not removed (at least no denitrification).
Response 2: My analysis is as follows: After testing, the total nitrogen is slightly higher than the ammonia nitrogen, indicating that the concentrations of NO3- and NO2- in the sewage are very low, and the total nitrogen content is equal to the ammonia nitrogen content, indicating that denitrification has indeed taken place, which can prove thatN2 was generated and denitrification was completed.
Comment 3: Also, as the authors suggested, glucose or succinate was needed as a carbon source during the process, this indicated there was limited economic value of applying the synthetic community in wastewater treatment.
Response 3: The experimental results show that sodium succinate is indeed the best carbon source, but the gap between sodium acetate and sodium acetate is only 0.76%, and the gap is limited. In the subsequent actual production, sodium acetate can be selected as the carbon source. At present, the types of carbon sources tested in experiments are limited, and other carbon sources can be further researched from an economic perspective.
Comment 4: Line 2, the title, a mixed culture can’t be called as ‘composite strain’, better to use ‘synthetic microbial community’ throughout the manuscript;
Response 4: The wording in the text has been modified, and "the synthetic microbial community" better reflects the purpose of experimental design.
Comment 5: Line 15, For wastewater treatment, only one strain was used is hardly true.
Response 5: The original intention is to express much-related research on a single strain, which has been modified.
Comment 6: All bacterial names need to be in italic
Response 6: All bacteria names have been changed to italics.
Comment 7: Line38-40, the meaning is unclear, please check the grammar
Response 7: The Sentences have been modified to “The COD and total nitrogen from aquaculture wastewater account for 96% and 38% of total agricultural wastewater, respectively.”
Comment 8: Line73, change to ‘higher removal efficiency of nitrogen and COD and less nitrous oxide…’
Response 8: The Sentences have been modified
Comment 9: Line 116, what’s the logic of this sentence?
Response 9: The title of the section means to screen all the strains isolated from the sludge and to identify the better-performing strains.
Comment 10: Line 133, the primer is 1492R
Response 10: The typing error has been modified.
Comment 11: Figure 1-4, it is meaningless to show the plate images
Response 11: The plate images have been deleted, and the picture numbers have been adjusted.
Comment 12: Fgiure5, what does the author want to show here? I suggest to substitute the phylogeny with a table indicating to which known bacteria each isolate was closely related, and the identity between their 16S rRNA sequences.
Response 12: In related strain experiments, the results of strain identification generally take the form of phylogenetic tree display, which is a method of describing the correlation between different organisms in bioinformatics. Systematic classification analysis can help people understand the evolutionary history of all living things.
Special thanks to you for your good comments. If you have any questions to discuss, please contact us.
Yours,
Co-authors.
Author Response File: 
Author Response.docx
Reviewer 2 Report
General comments:
This is a well-written, interesting paper researching an important and hot topic: heterotrophic nitrification-aerobic denitrification. There is a lot of good general information in the paper about the strains tested. However, the authors need to discussion the underlying mechanisms of why phenomena occur. For example in Section 3.3, the reader isn't provided with information about why potassium sodium tartrate was chosen as a carbon source (it seems unusual) and why it performed so poorly compared with the other substrates. I know it's probably self explanatory but it would be nice to have it spelled out. Is there a limiting concentration for any of these carbon sources before preferred nitrogen sources change or inhibition occurs?
Here are more specifics:
What was the dissolved oxygen (DO) concentrations in your different scenarios? I think that DO concentration would affect the efficacy and rate of different pathways. If any oxygen is present, it needs to be discussed in all of the results sections. What about the formation of intermediates like NO and N2O? Part of the controversy with this topic area is the release of potent greenhouse gases. Why were these not included? It would be useful to the reader to have the kinetics translated to maximum utilization rates and half saturation concentrations for easy comparison between the observed pathways and with other works. For Section 3.4: You report NH4+ removal, but was the nitrogen transformed to nitrate or nitrite or other intermediates? You also observed a decrease in NH4+ removal at a C/N ratio greater than 10. Could the heterotrophs be more likely to revert to aerobic respiration at the higher C/N ratio? For Section 3.7: I feel like more conclusions can be drawn from this data to determine if all of the right composite ratios were analyzed. Why did you use higher ratios of Acinetobacter, since is seems to be almost detrimental to the system. It almost seems like reducing the overall ratio of Pseudomonas in the system was as detrimental as higher concentrations of Acinetobacter. There's just more to look at with these relationships. When you varied the composite ratios, did you see changes in the community composition before and after the experiments? For Section 3.8: I am confused as to why NH4+ removal suddenly increased from the reactor on 20 Sept. Something must have significantly changed in the system. Please look into this.Minor comments:
Lines 105 & 107: Please state what the acronyms "BTB" and "LB" are. Line 182: Can you add a sentence or two as to why you chose these composite strain ratios? Line 266: I believe the "KO3-" should actually be either "KNO3" or "NO3-", I'm not sure which but I think either would do. Figure 9: The graphs are out of order. Please fix. Line 333: I think you mean to say that the composition strain demonstrated acid and alkali inhibition, not resistance, at pH 5 and 9. Is that correct?Author Response
We appreciate your careful review and review of our manuscript. Your comments are significant for the improvement and condensing of our manuscript. Moreover, your comments also point out a lot of interesting research directions, which provides a reference for us to carry out further research in the future. We have studied comments carefully and have made corrections and responses, which we hope to meet with approval. Revised portions are marked in yellow in the paper. The primary corrections in the paper and the responses to the reviewer’s comments are as following:
Comment 1: For example in Section 3.3, the reader isn't provided with information about why potassium sodium tartrate was chosen as a carbon source (it seems unusual) and why it performed so poorly compared with the other substrates. I know it's probably self-explanatory but it would be nice to have it spelled out.
Response 1: The performance of sodium tartrate is reduced because the carbon-to-nitrogen ratio of different carbon sources is consistent in our experiments, which leads to the consistency of the carbon source concentration, and the reaction process using sodium tartrate as a carbon source generally requires higher The concentration of the carbon source, therefore, reflects the effect deviation.
Comment 2: Is there a limiting concentration for any of these carbon sources before preferred nitrogen sources change or inhibition occurs?
Response 2: The concentration for any of these carbon sources was（C/N）1：10.
Comment 3: What was the dissolved oxygen (DO) concentrations in your different scenarios? I think that DO concentration would affect the efficacy and rate of different pathways. If any oxygen is present, it needs to be discussed in all of the results sections.
Response 3: The range of the DO concentration threshold is relatively broad and is not clearly defined. In this experiment, the concentration of DO in the feed water was controlled on average to about 1.0 mg / L to control its stability. The DO value has been added to the manuscript.
Comment 4: What about the formation of intermediates like NO and N2O? Part of the controversy with this topic area is the release of potent greenhouse gases. Why were these not included? It would be useful to the reader to have the kinetics translated to maximum utilization rates and half-saturation concentrations for easy comparison between the observed pathways and with other works.
Response 4: The analysis of the results of this experiment is aimed at the removal efficiency of ammonia nitrogen in wastewater. Due to limited time, NO and N2O greenhouse gases have not been measured. The next step is to hope to carry out research in this area.
Comment 5: For Section 3.4: You report NH4+ removal, but was the nitrogen transformed to nitrate or nitrite or other intermediates? You also observed a decrease in NH4+ removal at a C/N ratio greater than 10. Could the heterotrophs be more likely to revert to aerobic respiration at the higher C/N ratio?
Response 5: In section 3.2, we can see that when NH4Cl is fixed as the sole and only nitrogen source, the amount of NO2- and NO3- produced is very small, so there is no repeated detection in subsequent experiments. The higher carbon-nitrogen ratio makes the amount of carbon source too high, and some organics will directly embed the enzyme structure and affect the enzyme activity (Song Yujie et al., 2013), inhibiting the growth and nitrification of the strain.
Comment 6: For Section 3.7: I feel like more conclusions can be drawn from this data to determine if all of the right composite ratios were analyzed. Why did you use higher ratios of Acinetobacter, since is seems to be almost detrimental to the system. It almost seems like reducing the overall ratio of Pseudomonas in the system was as detrimental as higher concentrations of Acinetobacter. There's just more to look at with these relationships. When you varied the composite ratios, did you see changes in the community composition before and after the experiments?
Response 6: Comparing experimental group 1 and experimental group 2, experimental group 8 and experimental group 9, a single increase of the ratio of Acinetobacter sp. or a single decrease of the ratio of Pseudomonas sp. is harmful to the entire system. However, from the perspective of the interaction between microorganisms, it is suitable for the whole system to reduce the ratio of Pseudomonas sp. And the ratio of Bacillus sp. Furthermore, increase Acinetobacter sp. And Sphingobacterium sp. When changing the ratio, we did not micrograph the colonies. And it was regrettable that when changing the ratio, we did not micrograph the colonies.
Comment 7: For Section 3.8: I am confused as to why NH4+ removal suddenly increased from the reactor on 20 Sept. Something must have significantly changed in the system.
Response 7: Due to the rain, the effluent is diluted, and the ammonia nitrogen in the inlet water is lower than usual. According to the analysis, the excessively high ammonia nitrogen concentration in the inlet water may not be suitable for microbial reactions. After the ammonia nitrogen concentration is appropriately reduced, the removal rate is significantly increased.
Comment 8: Lines 105 & 107: Please state what the acronyms "BTB" and "LB" are.
Response 8: The BTB medium is the bromothymol blue medium and the LB medium is the Luria-Bertani medium.
Comment 9: Line 182: Can you add a sentence or two as to why you chose these composite strain ratios?
Response 9: In the pre-experiment, we tested the nitrification ability of different strains. The adjusted ratio is designed based on its removal performance.
Comment 10: Line 266: I believe the "KO3-" should actually be either "KNO3" or "NO3-", I'm not sure which but I think either would do.
Response 10: The original text has been modified to NO3-.
Comment 11: Figure 9: The graphs are out of order. Please fix.
Response 11: The picture order has been revised.
Comment 12: Line 333: I think you mean to say that the composition strain demonstrated acid and alkali inhibition, not resistance, at pH 5 and 9. Is that correct?
Response 12: Yes, the original text has been modified.
Special thanks to you for your good comments. If you have any questions to discuss, please contact us.
Yours,
Co-authors.
Author Response File: Author Response.docx
Reviewer 3 Report
The paper is devoted to an interesting and relevant topic - heterotrophic nitrification-aerobic denitrification as a new type of biological nitrogen removal technology. Compared with traditional biological nitrogen removal, the heterotrophic nitrification-aerobic denitrification process has a higher nitrogen removal efficiency and less nitrous oxide production. The paper certainly must be published, but there are some minor comments.
Lines: 23, 25, 37 79. Abbreviations are presented without preliminary decoding (SBR, COD). Decoding of the abbreviation COD is given only on line 37, although it is first encountered in the abstract on line 25.
Lines 113-114. The pressure at which sterilization was carried out is not indicated.
In the legend of Figure 6, nitrates are indicated. However, they are not in the Figure.
Figure 9 and its description in paragraph 3.6 are uninformative, since the temperature practically does not affect the process. They may be removed from the manuscript.
Figure 10 is difficult to understand without Table 1. May it be transferred to the table?
A detailed explanation is required of why, in Figure 12, after September 20, the value of COD sharply differs before and after the reactor, and until that time, no.
Author Response
Dear Reviewer:
We appreciate your careful review and review of our manuscript. Your comments are significant for the improvement and condensing of our manuscript. Moreover, your comments also point out a lot of interesting research directions, which provides a reference for us to carry out further research in the future. We have studied comments carefully and have made corrections and responses, which we hope to meet with approval. Revised portions are marked in yellow in the paper. The primary corrections in the paper and the responses to the reviewer’s comments are as following:
Comment 1: Lines: 23, 25, 37 79. Abbreviations are presented without preliminary decoding (SBR, COD). Decoding of the abbreviation COD is given only on line 37, although it is first encountered in the abstract on line 25.
Response 1: The first abbreviations were spelled out completely.
Comment 2: Lines 113-114. The pressure at which sterilization was carried out is not indicated.
Response 2: The value of the sterilized pressure was supplemented.
Comment 3: In the legend of Figure 6, nitrates are indicated. However, they are not in the Figure.
Response 3: In fact, there are. After carefully zooming in, you can see that the vertical axis value of the curve of nitrates is close to 0, and it is covered with the NO2- curve.
Comment 4: Figure 9 and its description in paragraph 3.6 are uninformative since the temperature practically does not affect the process. They may be removed from the manuscript.
Response 4: From the results, the change in temperature from 20-40 ° C is not very obvious, but it is also meaningful. For example, we do not need to reach high temperatures, which will not affect the reaction process and save costs.
Comment 5: Figure 10 is difficult to understand without Table 1. May it be transferred to the table?
Response 5: The figure 10 has been converted to a table.
Comment 6: A detailed explanation is required of why, in Figure 12, after September 20, the value of COD sharply differs before and after the reactor, and until that time, no.
Response 6: Due to the rain, the effluent is diluted and the ammonia nitrogen in the inlet water is lower than usual. According to the analysis, the excessively high ammonia nitrogen concentration in the inlet water may not be suitable for microbial reactions. After the ammonia nitrogen concentration is appropriately reduced, the removal rate is significantly increased.
Special thanks to you for your good comments. If you have any questions to discuss, please contact us.
Yours,
Co-authors.
Author Response File: Author Response.docx
Round 2
Reviewer 2 Report
The authors sufficiently addressed my general concerns. A couple of comments:
Please incorporate response #6 into your text to provide context on why certain ratios were not chosen. Please add in a sentence or two about the drop in NH4+ on Sept. 20.Nice paper!
Author Response
Dear reviewer:
Thanks again for your careful review and your comments are very meaningful for the details of the manuscript
Comment 1: Please incorporate response #6 into your text to provide context on why certain ratios were not chosen.
Response 1: In section 3.7, response #6 has been added to the text as a reason to explain the choice of matching. And the response #6 has been modified and reads as follows: Comparing experimental group 1 and experimental group 2, experimental group 8 and experimental group 9, a single increase of the ratio of Acinetobacter sp. or a single decrease of the ratio of Pseudomonas sp. is harmful to the entire system. However, from the perspective of the interaction between microorganisms, it is suitable for the whole system to reduce the ratio of Pseudomonas sp. and Bacillus sp. and increase the ratio of Acinetobacter sp. and Sphingobacterium sp. simultaneously.
Comment 2: Please add in a sentence or two about the drop in NH4+ on Sept. 20.
Response 2: In section 3.8, a few sentences have been added to explain the cause of the concentration of NH4+ reduction and analyze the changes in removal rate. And the added part is modified as follows: It was worth mentioning that due to the rain, the effluent was diluted, and the ammonia nitrogen in the inlet water was lower than usual. According to the analysis, the excessively high ammonia nitrogen concentration in the inlet water may not be suitable for microbial reactions. After the ammonia nitrogen concentration was appropriately reduced, the removal rate was significantly increased.
Special thanks to you for your good comments. If you have any questions to discuss, please contact us.
Yours,
Co-authors.
Author Response File: Author Response.docx
