Pollutant-Removing Biofilter Strains Associated with High Ammonia and Hydrogen Sulfide Removal Rate in a Livestock Wastewater Treatment Facility

Round 1

Reviewer 1 Report

The manuscript describes bacterial composition of wastewater within and after a biotreatment implementation, with the enumeration of species of bacteria useful for removing ammonia and hydrogen sulfide. They are using MiSeq on rDNA, and there is an extensive data analysis detailing different strain distributions as samples are taken in different times and different places in the treatment process. They also measured the efficiency of hydrogen sulfide and ammonia removal to correlate with the bacterial sampling. This is offered as a demonstration that MiSeq can provide stronger characterization of how the treatment operation is proceeding. The major problem is that the engineering details of the treatment operation are hard to make out, and there are no hypotheses stated. So the manuscript is an exercise in a statistically powerful measurement applied to a poorly defined question.

 

The engineering details of the filter: There is a diagram that implies the waste water is apparently cyclically sprinkled over some solid matrix, to which I assume the reactor bacteria to supposed to be adhered as biofilm. There is an input from a microbial supply tank shown to be injected somehow into the middle of this. What's in the supply tank is never described. Also, whether there is continuous inoculation, or just an initial inoculation, or something in between is never stated. The NFL sample is said to be taken from near the input of the effluent. Is it from the influent or from the NFL pond into which the sprinkled wastewater is cyclically draining? Is there some structure to the NFL pond to segment water based on number of passes through the filter, or is it all just mixed together? Is there something being done to keep the bacteria from the supply tank from intermixing in the NFL, or is it just mixed with the influent in hopes it will come to dominate the biofilm. Is the NFL sample supposed to monitor the state of the reactor bacteria or of the influent? Is bacterial supposed to adhere and stay resident in the filter, and if so has any sample of the biofilm been taken? Is the influent sludge with high bacterial content, or has it been mostly cleaned up prior to this step? Basically there are at least four bacterial populations here: the supply tank, the influent, the waste water possibly segregated by filtering stage, and the bacterial biofilm retained in the filter. What exactly is being sampled, and what exchanges occur among these populations is obtuse as described. Is there an assumption that bacteria in the NFL chamber is mainly leached from the biofilm in the filter?

 

I see a gas input; is the filter anoxic or aerated? There is talk of efficiency differences in summer and winter. Is the temperature maintained the same in the filter in summer and winter, or is it operated at ambient temperature, or something in between? If not temperature, what is proposed to cause these differences?

 

You say that species richness is higher in the effluent than in the NFL. How could that be possible? Is it because the NFL sample is mainly the influent bacteria and the effluent additionally contains bacteria derived from the microbial supply tank? Or is it that yet another population of bacteria exists in the way of biofilm accumulating in the NL chamber which is different because conditions are different in there, and some of that leaches into the effluent?

 

I certainly believe that Miseq gives a broader census of bacterial species present with far less effort than culturing. It is true that number of MiSeq reads can be used as a measure of abundance. This is mainly used to drive a diversity calculation which focuses on the relative abundance of the dominant species. It was also used to claim that there was a different total abundance of bacteria in the winter than the summer. For that purpose the samples should have been doped with a fixed amount of control bacteria (or cloned rDNA) to normalize for efficiency differences in the two MiSeq determinations. Take that for future reference.

 

For context, I'm a molecular biologist, and not a specialist in waste water management.

I'm not looking for anything to be done over or analyzed differently. For me to recommend publication to the editor, I need a better description of the experiment so I can imagine how the measurements fit into the operation of the plant, and have some understanding of what influences on the bacterial population you might be capturing.

Author Response

Q1.

The manuscript describes bacterial composition of wastewater within and after a biotreatment implementation, with the enumeration of species of bacteria useful for removing ammonia and hydrogen sulfide. They are using MiSeq on rDNA, and there is an extensive data analysis detailing different strain distributions as samples are taken in different times and different places in the treatment process. They also measured the efficiency of hydrogen sulfide and ammonia removal to correlate with the bacterial sampling. This is offered as a demonstration that MiSeq can provide stronger characterization of how the treatment operation is proceeding. The major problem is that the engineering details of the treatment operation are hard to make out, and there are no hypotheses stated. So the manuscript is an exercise in a statistically powerful measurement applied to a poorly defined question.

Response: We thank the reviewer for the suggestions that have further advanced our research. The overall study results have been revised and supplemented. Please check the manuscript text.

Q2.

The engineering details of the filter: There is a diagram that implies the waste water is apparently cyclically sprinkled over some solid matrix, to which I assume the reactor bacteria to supposed to be adhered as biofilm. There is an input from a microbial supply tank shown to be injected somehow into the middle of this. What's in the supply tank is never described. Also, whether there is continuous inoculation, or just an initial inoculation, or something in between is never stated. The NFL sample is said to be taken from near the input of the effluent. Is it from the influent or from the NFL pond into which the sprinkled wastewater is cyclically draining? Is there some structure to the NFL pond to segment water based on number of passes through the filter, or is it all just mixed together? Is there something being done to keep the bacteria from the supply tank from intermixing in the NFL, or is it just mixed with the influent in hopes it will come to dominate the biofilm. Is the NFL sample supposed to monitor the state of the reactor bacteria or of the influent? Is bacterial supposed to adhere and stay resident in the filter, and if so has any sample of the biofilm been taken? Is the influent sludge with high bacterial content, or has it been mostly cleaned up prior to this step? Basically there are at least four bacterial populations here: the supply tank, the influent, the waste water possibly segregated by filtering stage, and the bacterial biofilm retained in the filter. What exactly is being sampled, and what exchanges occur among these populations is obtuse as described. Is there an assumption that bacteria in the NFL chamber is mainly leached from the biofilm in the filter?

Response: We thank the reviewer for raising this crucial question. The microbial supply tank of the wastewater treatment facility that was investigated was a kind of microbial culture tank for wastewater treatment. Microbial culture materials, including organic carbon sources were supplied to the tank, and the cultured microorganisms were inoculated into the wastewater treatment facilities. The cultured microbes in the microbial supply tank were periodically inoculated into the filter area at regular intervals. The collected NLF samples were obtained from some of the treated wastewater filters. Biofilms formed on the surface of the filter, and the wastewater influent sprayed on the filter were included on the surface of the biofilms. Therefore, the collected NLF sample contained a mixture of biofilms that adhered to the filter surface and wastewater influent freshly sprayed on the filter.

Q3.

I see a gas input; is the filter anoxic or aerated? There is talk of efficiency differences in summer and winter. Is the temperature maintained the same in the filter in summer and winter, or is it operated at ambient temperature, or something in between? If not temperature, what is proposed to cause these differences?

Response: We thank the reviewer for asking this crucial question. The investigated wastewater treatment facility aimed not at removing contaminants through anaerobic bacteria. The facility is equipped with a device that introduces external air into the facility, and “Gas in let” in Figure 1 plays that role. Due to the absence of special heating and cooling systems in the facility, it was affected by seasonal temperature changes. Therefore, due to the characteristics of wastewater treatment facilities, changes in the external environment, including seasons, may affect the pollutant removal efficiency, and we suggest that these changes result from reflecting changes in the microbial community.

Q4.

You say that species richness is higher in the effluent than in the NFL. How could that be possible? Is it because the NFL sample is mainly the influent bacteria and the effluent additionally contains bacteria derived from the microbial supply tank? Or is it that yet another population of bacteria exists in the way of biofilm accumulating in the NL chamber which is different because conditions are different in there, and some of that leaches into the effluent?

Response: We thank the reviewer for raising this important question. Assuming that there is an early microbial community mixed with several species in an enclosed space, the species richness will be reduced due to the dominance and extinction of some species. However, we are interested in the fact that for the investigated wastewater treatment plant, the value of species richness in the NL presented at a later stage of the NLF was increased. We have established several hypotheses as to the cause of these results. One hypothesis states that the number of cells in the NLF sample was low enough to be difficult to detect by Illumina Miseq analysis, but the number of cells in the NL proliferated to a detectable level. We found that as the number of validated reads identified was higher in the NL than in the NLF, a species that was difficult to detect in the NLF was likely to be detected in the NL. According to our experience, repeated analysis of DNA extracted from the same sample expressed similar values in the dominance rate of the dominant species, but it was not always detected in species with few cells. Therefore, some species may not have been accidentally included in the results of the NLF. However, it is insufficient to explain the difference in species richness between NLF and NL through undetected species. Another hypothesis could be added; the composition of the inoculated microbial group provided from the microbial supply tank may have changed over time. Regarding the filter directly affected by the microbial group provided from the microbial supply tank, it will depend on the inoculated microbial group, but long-term changes in the microbial community may accumulate in the wastewater passed through the filter.

Q5.

I certainly believe that Miseq gives a broader census of bacterial species present with far less effort than culturing. It is true that number of MiSeq reads can be used as a measure of abundance. This is mainly used to drive a diversity calculation which focuses on the relative abundance of the dominant species. It was also used to claim that there was a different total abundance of bacteria in the winter than the summer. For that purpose the samples should have been doped with a fixed amount of control bacteria (or cloned rDNA) to normalize for efficiency differences in the two MiSeq determinations. Take that for future reference.

Response: We thank the reviewer for making this vital suggestion. We agree with the reviewer’s opinion. We believe that the concentration of extracted DNA varies depending on the density of microbes present in the sample. The result reflected in the number of Miseq reads. Therefore, we support that the number of Miseq reads may indirectly indicate the scale of the microbial community. To support this claim, all procedures of the experiment were conducted under similar conditions, starting with the equal volume of the sample from which the DNA was extracted. We will refer to the reviewer’s suggestion for further study.

Q6.

For context, I'm a molecular biologist, and not a specialist in waste water management.

I'm not looking for anything to be done over or analyzed differently. For me to recommend publication to the editor, I need a better description of the experiment so I can imagine how the measurements fit into the operation of the plant, and have some understanding of what influences on the bacterial population you might be capturing.

Response: We thank the reviewer for the suggestions. (see below page 1 lines 39–42, page 2 lines 61-65 and lines 86-93, page 8 lines 351-354):

 

Wastewater treatment is an essential process directly linked to human health and environmental issues [1]. The construction of wastewater treatment systems and the development of facilities and technologies have protected humans from water-borne diseases [2].

To remove pollutants in wastewater, nitrogen and phosphorus-related chemicals were the primary evaluation criteria [15]. For pollutant removal meeting the evaluation criteria, various microbes, including denitrifying bacteria and microalgae, were used, and nitrogen and phosphorus could be successfully isolated from wastewater [16, 17]. However, despite the successful removal of nitrogen and phosphorus, the odor was uncontrollable due to odor-causing substances in some cases [18]. There are various odor-causing substances, and the types of microbes that decompose them are limited [19, 20].

A facility with a biofilter capable of removing contaminants, including VFAs, ammonia, and hydrogen sulfide, from various sewage was devised (Figure 1). To verify the effectiveness of this devised facility, the pollutant removal function needs to be evaluated, and a deep knowledge of the pollutant removal function requires an understanding of the microbial community. Therefore, in this study, the efficiency of the removal of odor-causing substances by livestock wastewater treatment facility was evaluated. Illumina MiSeq sequencing was used to reveal the relationship between the functionality of the biofilter system and the microbial community.

It is desirable to maintain the pollutant purification ability by constantly maintaining a certain functional microbial species, but considering this phenomenon, the most effective direction is to maintain the functionality by being replaced by a microbial species with similar function.

 

Reviewer 2 Report

The manuscript needs improvement. Please see specific comments below:

1) Only data at genus level should be performed.

2) The abundance of the major 25 genera should be placed in a bar chart.

3) Introduction should be expanded.

4) Diversity indices should be given.

5) A in depth discussion should be performed.

Author Response

Q1.

1) Only data at genus level should be performed.

Response: Following the reviewer’s suggestion, the manuscript was updated by adding the statement (see page 6 lines 247–261):

At the genus level, ten genera (Flavobacteriaceae, Alcaligenaceae, Pseudogracilibacillus, Limnochordaceae, Oleiphilaceae, Sphingobacterium, Guyparkeria, Moheibacter, Thioalkalibacteraceae, Rubricoccaceae) with a relative abundance of > 5% were identified. The sum of the relative abundances of these 10 genera was > 50% in all samples analyzed in June 2019 (NLF: 65.38%; NL: 58.17%) and January 2020 (NLF: 76.96%; NL: 75.64%). For the samples from June 2019, the microbial community of NLF was dominated by Oleiphilaceae (40.89%) and Thioalkalibacteraceae (12.91%), and the microbial community of NL was dominated by Limnochordaceae (41.83%), Moheibacter (9.67%), and Rubricocccaceae (5.50%). For samples from January 2020, the microbial communities of NLF and NL were dominated by Flavobacteriaceae (NLF: 23.83%; NL: 23.59%), Alcaligenaceae (NLF: 22.48%; NL: 21.75%), Pseudogracilibacillus (NLF: 19.01%; NL: 18.26%), Sphingobacterium (NLF: 5.44%; NL: 5.99%), and Guyparkeria (NLF: 5.96%; NL: 5.84%). In June 2019, Oleiphilaceae, Limnochordaceae, Moheibacter, Thioalkalibacteraceae, and Rubricoccaceae dominated, while in January 2020, Flavobacteriaceae, Alcaligenaceae, Pseudogracilibacillus, Sphingobacterium, and Guyparkeria dominated.

Q2.

2) The abundance of the major 25 genera should be placed in a bar chart.

Response: According to the reviewer’s suggestion, a bar chart was added in Figure 2 (b).

Figure 2. Taxonomic composition of the prokaryotic microbial phyla in the NLF and NL areas of the wastewater treatment facility shown in Figure 1. The relative abundance of bacterial (a) phyla and (b) genus are shown.

Q3.

3) Introduction should be expanded.

Response: Based on the reviewer’s suggestion, the manuscript was updated. (see pages 1-2 lines 39–96):

Wastewater treatment is an essential process that is directly linked to human health and environmental issues [1]. The construction of wastewater treatment systems and the development of facilities and technologies have protected humans from water-borne diseases [2]. Additionally, the destruction and pollution in aquatic ecosystem, such as eutrophication and toxic algal blooms, that may occur due to wastewater could be prevented through improved wastewater treatment [3]. With the development of industries, the amount of wastewater discharged by humanity has increased, and the types of wastewater have also diversified [4]. As the types of wastewater have diversified to keep pace with these changes, various approaches to wastewater treatment are required [4]. As such, it is vital to conduct research on efficient wastewater treatment [5], from which various wastewater treatment facilities and systems can be developed [5, 6]. Physical methods that depend on particle sizes, such as sedimentation or filtration, are introduced [7]. By such, facilities and systems, suspended solids and debris can be separated from wastewater [8]; however, there were limits to chemicals that could not be removed through physical filtration [9]. In wastewater, not only a large number of pollutants such as nitrogen and phosphorus compounds exist, but also chemicals that cause odor vary depending on the wastewater source [10, 11]. Therefore, it was necessary to supplement the removal of pollutants, including odor-causing chemicals [8, 12]. To remove pollutants that cannot be removed by physical filtration, a biological purification process was applied [12]; this process uses microorganisms capable of decomposing pollutants, including odor-causing substances [13], and it enables the decomposition of both odor-related compounds and some toxic substances [14].

To remove pollutants contained in wastewater, nitrogen, and phosphorus-related chemicals were the primary evaluation criteria [15]. For pollutant removal to meet the evaluation criteria, various microorganisms, including denitrifying bacteria and microalgae, were used, and nitrogen and phosphorus could be successfully isolated from wastewater [16, 17]. However, although the successful removal of nitrogen and phosphorus, odors were not controlled due to odor-causing substances in some cases [18]. There are various odor-causing substances, and the types of microorganisms that decompose them are limited [19, 20]. In wastewater, many kinds of odor-causing substances exist, including volatile fatty acids (VFAs), ammonia, and hydrogen sulfide [13, 21]; thus, various types of microbes are used to decompose and purify these substances [13, 22]. Previous studies have shown that several bacterial species of the genus Dietzia have the ability to decompose VFAs [23], while some bacterial species of Alcaligenaceae and Oleiphilaceae are effective in removing ammonia [24–26], and various bacterial strains belonging to genera, such as Comamonas, Guyparkeria, and Aquamicrobium or the family Thioalkalibacteraceae can be used to treat hydrogen sulfide [27–33]. These findings have shown the basis for applying functional microorganisms in wastewater treatment facilities [23, 25, 27, 33]. However, given that microbial communities can change because of environmental factors and seasonal changes, there remains a need for studies on microbial community characteristics and the functionality of single strains [34–36]. Many efforts to analyze the characteristics of the microbial community through metagenomic methods have been attempted in previous studies [37]. These attempts have filled the insurmountable limitations of functional identification of microbial communities through culture-based assays [37, 38].

In the Republic of Korea, wastewater treatment is becoming increasingly essential because of the increase in the number and size of livestock farms [39]. Ade-quate removal of odor-causing chemicals is a critical issue for treating wastewater from livestock farms [40]. Therefore, a facility with a biofilter capable of removing contaminants, including VFAs, ammonia, and hydrogen sulfide, from various wastewaters was devised (Figure 1). To verify the effectiveness of this devised fa-cility, the pollutant removal function needs to be evaluated, and a deep knowledge of the pollutant removal function requires an understanding of the microbial community. Therefore, in this study, the efficiency of the removal of odor-causing substances by livestock wastewater treatment facility was evaluated, Illumina MiSeq sequencing was used to reveal the relationship between the functionality of the biofilter system and the microbial community. Furthermore, this sequencing technique was used to compare the winter and summer microbial communities. In addition, through comparison with culture-based analysis, Illumina MiSeq sequencing was shown to be a superior method for microbial community analysis.

Q4.

4) Diversity indices should be given.

Response: We thank the reviewer for making this vital suggestion. To assess the diversity of the microbial community, Table 1 lists three indices indicating species richness and species diversity. Chao1 is an indicator of species richness, Shannon and Simpson are indicators of species diversity. These three indicators quantify the community’s diversity by reflecting information such as the number of species constituting the microbial community and the abundance of each species.

Q5.

5) A in depth discussion should be performed.

Response: Based on the reviewer’s suggestion, we have added the sentence (see pages 7–8 lines 327–354):

3.4. Relationship between Pollutant Purification and the Microbial Community

The investigated facility demonstrated that livestock wastewater ammonia and hydrogen sulfide could be effectively removed (Table 1). Additionally, the Illumina MiSeq result expressed that pollutant purification-related microbes existed in the biofilter (NLF) and treated effluent (NL) (Table 2). Interestingly, although ammonia and hydrogen sulfide were removed at both periods, June 2019 and January 2020, there were differences between the samples at both periods in the dominant species constituting the microbial community. The result supports that the purification capacity for ammonia and hydrogen sulfide can be maintained despite the changes in the species constituting the microbial community [68, 69]. Changes in microbial species composition are of great concern in open-type purification facilities using microbes [69, 70]. Due to the characteristic of the facility where microorganisms purify pollutants, the pollutant purification ability depends on the characteristics of the microorganisms. The facility’s utility can be determined by the characteristics of the microbial community [71, 72]. Therefore, these facilities are sensitive to changes in the microbial community and require strategies for maintenance and control of the microbial species that can provide the desired function [73, 74]. However, open-type facilities are vulnerable to the introduction of external microorganisms [75]. Furthermore, changes in wastewater properties, such as changes in organic carbon sources or the presence or absence of antibiotics, can change the composition of the microbial community [76]. Consequently, maintaining a particular species of microorganism is not easy. The reason wastewater treatment facilities can remain functional despite the difficulty in controlling changes in the microbial community is that microbial species with similar functions can replace the role of existing microbial species [69, 74]. Our findings are also expected to be one of the cases in which microbial species have replaced functional microbial species in the microbial community with similar roles. It is desirable to maintain the pollutant purification ability by constantly maintaining a certain functional microbial species, but considering this phenomenon, the most effective direction is to maintain the functionality by being replaced by a microbial species with similar function.

Reviewer 3 Report

The Authors should brefly describe the technological layout of the livestock wastewater treatment  plant and the quality of wastewater directed to it. The reason is that different compositions of wastewater and various  technological processes  generate  different  concentrations of ammonia, VFA and hydrogen sulphide.

Author Response

Q1.

The Authors should brefly describe the technological layout of the livestock wastewater treatment plant and the quality of wastewater directed to it. The reason is that different compositions of wastewater and various technological processes generate different concentrations of ammonia, VFA and hydrogen sulphide.

 

Response: We thank the reviewer for the suggestions made. However, the schematic diagram presented in Figure 1 contains all the operating principles of the facility. The whole process of facility operation is a cyclical process in which newly introduced wastewater is sprayed into the biofilter through pipes and pumps. When the wastewater pollutants were removed, it was discharged from the facility as the last process. No additional physicochemical factors were measured for the condition of wastewater entering the facility at the time of sample collection. The information on ammonia and hydrogen sulfide summarized in Table 1 is the only information that can estimate the quality of wastewater at that time.

 

In the Republic of Korea, wastewater treatment is becoming increasingly essential because of the increase in the number and size of livestock farms [39]. Ade-quate removal of odor-causing chemicals is a critical issue for treating wastewater from livestock farms [40]. Therefore, a facility with a biofilter capable of removing contaminants, including VFAs, ammonia, and hydrogen sulfide, from various sewage was devised (Figure 1). To verify the effectiveness of this devised facility, the pollutant removal function needs to be evaluated, and a deep knowledge of the pollutant removal function requires an under-standing of the microbial community. Therefore, in this study, the efficiency of the remov-al of odor-causing substances by livestock wastewater treatment facility was evaluated. Illumina MiSeq sequencing was used to reveal the relationship between the functionality of the biofilter system and the microbial community. Furthermore, this sequencing tech-nique was used to compare the winter and summer microbial communities. Additionally, through comparison with culture-based analysis, Illumina MiSeq sequencing was shown to be a superior method for microbial community analysis. (see page 2, lines 83–96).

Reviewer 4 Report

The paper entitled: „Pollutant-Removing Biofilter Strains Associated with High Ammonia and Hydrogen Sulfide-Removal Rate in a Livestock Wastewater Treatment Facility” deals with an experimental study aimed at evaluating the relationship between pollutant removal and the microbial community in a wastewater treatment facility. I think, that the manuscript requires extensive revision to improve the quality of presentation.

Language checking is also necessary.

It is an interesting paper, however a lot improvements are necessary:

• Abstract

 

Please do not write an abstract this way.

An abstract should be able to stand alone and independent of the paper.

Moreover, an abstract need to state more clearly aim of your work and methodological approach as well. Rephrase.

 

• Introduction

 

The introduction is not very strong. It is very poor in information. It lacks a review of previous research. It would need to identify what led to the design and selection of this segment of research and what was conducted in previous research that led to this. It would also be extremely important to emphasize the novel nature of the research.

Furthermore, the introduction is not the place to present the results of the study – that is what the results section (lines 63 – 65).

 

• Materials and Methods

 

Materials and Methods section are presented in an unreadable way. It should be reworded a bit and descriptions should be simplified.

I also suggest to divide it into sub- chapters.

Most importantly, what is missing here is a detailed full characterization of the wastewater collected from two areas.

Moreover, the authors do not explain why such research periods were chosen.

 

• Result and Discussion

 

The results of the study are presented in an unreadable way. They should be reworded a bit and descriptions should be simplified.

However, among other crucial problems is missing discussion. Discussion should unambiguously express a comparison of the achieved results with the previous knowledge of the topic. It must make clear what is completely new in the presented results and where these results differ from the findings of other authors, and in what they coincide with the published opinions. Discussion should emphasise to the newly opened issues and the need for their solution. This is completely missing.

 

• Conclusions

 

The reader should be provided with real conclusions at the end of the study. This section is not written as summary – but what are the conclusions from your research?

Author Response

Q1.

Please do not write an abstract this way.

An abstract should be able to stand alone and independent of the paper.

Moreover, an abstract need to state more clearly aim of your work and methodological approach as well. Rephrase.

 

Response: According to the reviewer’s suggestion, we updated the manuscript by rewriting some part of the abstract. (see page 1 lines 15–21 and lines 29–31):

This study analyzed the microbial community metagenomically to determine the cause of the functionality of a livestock wastewater treatment facility that can effectively remove pollutants, such as ammonia and hydrogen sulfide. Illumina MiSeq sequencing was used in analyzing the composition and structure of the microbial community, 16S rRNA gene was used. Through Illumina MiSeq sequencing, information such as diversity indicators as well as the composition and structure of microbial communities present in the livestock wastewater treatment facility were obtained, and differences between microbial communities present in the investigated samples were compared.

Therefore, the functionality of the livestock wastewater treatment facility was affected by characteristics, including the composition of the microbial community.

 

 

Q2.

The introduction is not very strong. It is very poor in information. It lacks a review of previous research. It would need to identify what led to the design and selection of this segment of research and what was conducted in previous research that led to this. It would also be extremely important to emphasize the novel nature of the research.

Furthermore, the introduction is not the place to present the results of the study – that is what the results section (lines 63 – 65).

 

Response: According to the reviewer’s suggestion, we updated the introduction as follows (see pages 1–2 lines 39–96):

Wastewater treatment is an essential process that is directly linked to human health and environmental issues [1]. The construction of wastewater treatment systems and the development of facilities and technologies have protected humans from water-borne diseases [2]. Additionally, the destruction and pollution in aquatic ecosystems, such as eutrophication and toxic algal blooms, that may occur due to wastewater could be prevented through improved wastewater treatment [3]. With the development of industry, the amount of wastewater discharged by humanity has increased, and the types of wastewater have also diversified [4]. As the types of wastewater have diversified to keep pace with these changes, various approaches to wastewater treatment are required [4]. As such, it is vital to conduct research on efficient wastewater treatment [5], from which various wastewater treatment facilities and systems can be developed [5, 6]. Physical methods that depend on particle sizes, such as sedimentation or filtration, are introduced [7]. By such facilities and systems, suspended solids and debris can be separated from wastewater [8]; however, there were limits to chemicals that could not be removed through physical filtration [9]. In wastewater, not only a large number of pollutants such as nitrogen and phosphorus compounds exist, but also chemicals that cause odor vary depending on the wastewater source [10, 11]. Therefore, it was necessary to supplement the removal of pollutants, including odor-causing chemicals [8, 12]. To remove pollutants that cannot be removed by physical filtration, a biological purification process was applied [12]; this process uses microbes capable of decomposing pollutants, including odor-causing substances [13], and it enables the decomposition of both odor-related compounds and some toxic substances [14].

To remove pollutants contained in wastewater, nitrogen and phosphorus-related chemicals were the primary evaluation criteria [15]. For pollutant removal to meet the evaluation criteria, various microorganisms, including denitrifying bacteria and microalgae, were utilized, and nitrogen and phosphorus could be isolated from the wastewater [16, 17]. However, although the successful removal of nitrogen and phosphorus, odors were not controlled due to odor-causing substances in some cases [18]. There are various odor-causing substances, and the types of microorganisms that decompose them are limited [19, 20]. In wastewater, many types of odor-causing substances exist, including VFAs, ammonia, and hydrogen sulfide [13, 21]; thus, various types of microorganisms are used to decompose and purify these substances [13, 22]. Previous studies have shown that several bacterial species of the genus Dietzia have the ability to decompose VFAs [23], while some bacterial species of Alcaligenaceae and Oleiphilaceae are effective in removing ammonia [24–26], and various bacterial strains belonging to genera, such as Comamonas, Guyparkeria, and Aquamicrobium or the family Thioalkalibacteraceae can be used to treat hydrogen sulfide [27–33]. These findings have shown the basis for applying functional microorganisms in wastewater treatment facilities [23, 25, 27, 33]. However, given that microbial communities can change because of environmental factors and seasonal changes, there remains a need for studies on microbial community characteristics and the functionality of single strains [34–36]. Many efforts to analyze the characteristics of the microbial community through metagenomic methods have been attempted by previous research [37]. These attempts have filled the insurmountable limitations of functional identification of microbial communities through culture-based assays [37, 38].

In the Republic of Korea, wastewater treatment is becoming increasingly essential because of the increase in the number and size of livestock farms [39]. Ade-quate removal of odor-causing chemicals is a critical issue for treating wastewater from livestock farms [40]. Therefore, a facility with a biofilter capable of removing contaminants, including VFAs, ammonia, and hydrogen sulfide, from various sewage was devised (Figure 1). To verify the effectiveness of this devised facility, the pollutant removal function needs to be evaluated, and a deep knowledge of the pollutant removal function requires an understanding of the microbial community. Therefore, in this study, the efficiency of the removal of odor-causing substances by livestock wastewater treatment facility was evaluated. Illumina MiSeq sequencing was used to reveal the relationship between the functionality of the biofilter system and the microbial community. Furthermore, this sequencing technique was used to compare the winter and summer microbial communities. Additionally, through comparison with culture-based analysis, Illumina MiSeq sequencing was shown to be a superior method for microbial community analysis.

Q3.

Materials and Methods section are presented in an unreadable way. It should be reworded a bit and descriptions should be simplified.

I also suggest to divide it into sub- chapters.

Most importantly, what is missing here is a detailed full characterization of the wastewater collected from two areas.

Moreover, the authors do not explain why such research periods were chosen.

 

Response: Based on the reviewer’s suggestion, we updated the manuscript. (see lines 98–101, lines 114–117, and lines 128–136):

2.1. Measurement of Ammonia

The concentrations of ammonia were measured to confirm the ability of the wastewater treatment facility to remove pollutants; for this purpose, the methods for the spectrophotometric determination of ammonia were used [25, 26].

2.2. Measurement of Sulfur Compounds

The concentrations of sulfur compounds were measured to confirm the ability of the wastewater treatment facility to remove pollutants; for this purpose, the packed gas chromatography methods were used.

NLF samples were collected from the biofilter of the investigated plant. The NLF samples included wastewater introduced into the plant, microbial culture from the microbial supply tank, and a biofilm formed on the surface of the biofilter. NL samples were collected at the discharge stage of the investigated plant after the wastewater treatment process was completed. The NL samples included only effluents that had undergone wastewater treatment with biofilters. Each sample was collected at 1 L per site. Samples were collected in June 2019 and January 2020 to identify changes and differences in the microbial community with seasonal and temporal changes.

Q4.

The results of the study are presented in an unreadable way. They should be reworded a bit and descriptions should be simplified.

However, among other crucial problems is missing discussion. Discussion should unambiguously express a comparison of the achieved results with the previous knowledge of the topic. It must make clear what is completely new in the presented results and where these results differ from the findings of other authors, and in what they coincide with the published opinions. Discussion should emphasise to the newly opened issues and the need for their solution. This is completely missing.

 

Response: Based on the reviewer’s suggestion, we updated the manuscript. (see pages 7–8, lines 327–354):

3.4. Relationship between Pollutant Purification and the Microbial Community

The investigated facility demonstrated that livestock wastewater ammonia and hydrogen sulfide could be effectively removed (Table 1). Additionally, the Illumina MiSeq result showed that pollutant purification-related microorganisms existed in the biofilter (NLF) and treated effluent (NL) (Table 2). Although ammo-nia and hydrogen sulfide were removed at both periods, June 2019 and January 2020, there were differences between the samples at both periods in the dominant species constituting the microbial community. These results support that the purification capacity for ammonia and hydrogen sulfide can be maintained despite the changes in the species constituting the microbial community [68, 69]. Changes in microbial species composition are of great concern in open-type purification facilities using microorganisms [69, 70]. Due to the characteristic of the facility where microorganisms purify pollutants, the pollutant purification ability depends on the characteristics of the microorganisms, and the utility of the facility can be determined by the characteristics of the microbial community [71, 72]. Therefore, these types of facilities are sensitive to changes in the microbial community and require strategies to maintain and control the microbial species that can provide the desired function [73, 74]. However, open-type facilities are vulnerable to the introduction of external microorganisms [75]. Furthermore, changes in wastewater properties, such as changes in organic carbon sources or the presence or absence of antibiotics, can change the composition of the microbial community [76]. Consequently, maintaining a particular species of microorganism is difficult. The reason wastewater treatment facilities can remain functional despite the difficulty in controlling changes in the microbial community is that microbial species with similar functions can replace existing microbial species [69, 74]. Our findings are also expected to be one of the cases in which functional microbial species in the microbial community have been replaced by microbial species with similar roles. It is desirable to maintain the pollutant purification ability by constantly maintaining a certain functional microbial species, but considering this phenomenon, the most effective direction is to maintain the functionality replacing it with microbial species of similar function.

Q5.

The reader should be provided with real conclusions at the end of the study. This section is not written as summary – but what are the conclusions from your research?

 

Response: According to the reviewer’s suggestion, we have added the sentence (see page 8, lines 365–367):

In conclusion, the effective pollutant removal of wastewater treatment facility depends on the microbial community of the biofilter, and a broad understanding of the microbial community can help effective wastewater treatment.

Round 2

Reviewer 1 Report

I have no further comments.

Author Response

Response to Reviewers’ Comments

 

Response to Editors’ comments (sky blue color )

Thank you very much for your e-mail on June 22, 2021.

We were pleased to hear that our manuscript “Pollutant-Removing Biofilter Strains Associated with High Ammonia and Hydrogen Sulfide-Removal Rate in Livestock Wastewater Treatment Facility” (Manuscript ID: sustainability-1214279) will be considered again for publication if appropriately revised. As such, please find attached the revised manuscript, with changes made in response to the suggestions of the reviewers marked in sky blue color (Editor).

 

 

Academic Editor Notes

 

 

 

Q1. There still some issues to address before acceptance for publication:

 

Response: We thank the editor for the suggestions, which have advanced our research. We revised and supplemented the overall study results. Please check the manuscript text.

 

 

Q2. - Abstract. NLF and NL should be defined in the abstract. If they are just influent and effluent samples, please use these terms.

 

Response: We thank the editor for raising this important suggestion. In abstract, NLF and NL were replaced with influent sample (NLF) and effluent sample (NL). (See page 1, lines 22–30).

 

 

Q3. - Section 2. Material and Methods. There is no information about the wastewater treatment process: aerobic/anaerobic combination process, activated sludge, trickling filter, membrane...? Please provide some information about the treatment type, daily flow treated...

 

Response: We thank the editor for raising this important suggestion. Based on the editor's suggestion, we added more details about the wastewater treatment process. The investigated wastewater treatment facility is equipped with a system that circulates wastewater through a pump to maintain aerobic conditions. Wastewater flows into the facility daily, and it has a step-by-step structure that can be spatially separated from previously treated wastewater. The newly introduced wastewater and the previously treated wastewater are exposed to the biofilter through the circulation process. A biofilter is a fixed-type solid-state filter, and the biofilter is sprayed with a microbial culture provided from a microbial supply tank. The system is built in such a way that the microbial community formed on the surface of the biofilter can be involved in the process of treating wastewater exposed to the biofilter. Wastewater that has passed through biofilter is evaluated for removal of pollutant before discharge, and wastewater from which pollutant has been removed is discharged from the facility. (See pages 3-4, lines 129–140).

 

 

Q4. - The English must be revised. Example: line 134: "Each sample was collected at 1 L per site"

 

Response: We thank the reviewer for pointing out these errors, and we have modified the text as follows (See page 4, lines 145–146): The 1 L bottle samples were collected at each site.

 

 

Reviewer 2 Report

The manuscript has been improved

Author Response

Response to Reviewers’ Comments

 

Response to Editors’ comments (sky blue color )

Thank you very much for your e-mail on June 22, 2021.

We were pleased to hear that our manuscript “Pollutant-Removing Biofilter Strains Associated with High Ammonia and Hydrogen Sulfide-Removal Rate in Livestock Wastewater Treatment Facility” (Manuscript ID: sustainability-1214279) will be considered again for publication if appropriately revised. As such, please find attached the revised manuscript, with changes made in response to the suggestions of the reviewers marked in sky blue color (Editor).

 

 

Academic Editor Notes

 

 

 

Q1. There still some issues to address before acceptance for publication:

 

Response: We thank the editor for the suggestions, which have advanced our research. We revised and supplemented the overall study results. Please check the manuscript text.

 

 

Q2. - Abstract. NLF and NL should be defined in the abstract. If they are just influent and effluent samples, please use these terms.

 

Response: We thank the editor for raising this important suggestion. In abstract, NLF and NL were replaced with influent sample (NLF) and effluent sample (NL). (See page 1, lines 22–30).

 

 

Q3. - Section 2. Material and Methods. There is no information about the wastewater treatment process: aerobic/anaerobic combination process, activated sludge, trickling filter, membrane...? Please provide some information about the treatment type, daily flow treated...

 

Response: We thank the editor for raising this important suggestion. Based on the editor's suggestion, we added more details about the wastewater treatment process. The investigated wastewater treatment facility is equipped with a system that circulates wastewater through a pump to maintain aerobic conditions. Wastewater flows into the facility daily, and it has a step-by-step structure that can be spatially separated from previously treated wastewater. The newly introduced wastewater and the previously treated wastewater are exposed to the biofilter through the circulation process. A biofilter is a fixed-type solid-state filter, and the biofilter is sprayed with a microbial culture provided from a microbial supply tank. The system is built in such a way that the microbial community formed on the surface of the biofilter can be involved in the process of treating wastewater exposed to the biofilter. Wastewater that has passed through biofilter is evaluated for removal of pollutant before discharge, and wastewater from which pollutant has been removed is discharged from the facility. (See pages 3-4, lines 129–140).

 

 

Q4. - The English must be revised. Example: line 134: "Each sample was collected at 1 L per site"

 

Response: We thank the reviewer for pointing out these errors, and we have modified the text as follows (See page 4, lines 145–146): The 1 L bottle samples were collected at each site.

 

 

Author Response File: Author Response.docx

Reviewer 4 Report

The authors have corrected the critical remarks suggested in the review. 
The paper is now in better condition than before - the corrections make difference.

Author Response

Response to Reviewers’ Comments

 

Response to Editors’ comments (sky blue color )

Thank you very much for your e-mail on June 22, 2021.

We were pleased to hear that our manuscript “Pollutant-Removing Biofilter Strains Associated with High Ammonia and Hydrogen Sulfide-Removal Rate in Livestock Wastewater Treatment Facility” (Manuscript ID: sustainability-1214279) will be considered again for publication if appropriately revised. As such, please find attached the revised manuscript, with changes made in response to the suggestions of the reviewers marked in sky blue color (Editor).

 

 

Academic Editor Notes

 

 

 

Q1. There still some issues to address before acceptance for publication:

 

Response: We thank the editor for the suggestions, which have advanced our research. We revised and supplemented the overall study results. Please check the manuscript text.

 

 

Q2. - Abstract. NLF and NL should be defined in the abstract. If they are just influent and effluent samples, please use these terms.

 

Response: We thank the editor for raising this important suggestion. In abstract, NLF and NL were replaced with influent sample (NLF) and effluent sample (NL). (See page 1, lines 22–30).

 

 

Q3. - Section 2. Material and Methods. There is no information about the wastewater treatment process: aerobic/anaerobic combination process, activated sludge, trickling filter, membrane...? Please provide some information about the treatment type, daily flow treated...

 

Response: We thank the editor for raising this important suggestion. Based on the editor's suggestion, we added more details about the wastewater treatment process. The investigated wastewater treatment facility is equipped with a system that circulates wastewater through a pump to maintain aerobic conditions. Wastewater flows into the facility daily, and it has a step-by-step structure that can be spatially separated from previously treated wastewater. The newly introduced wastewater and the previously treated wastewater are exposed to the biofilter through the circulation process. A biofilter is a fixed-type solid-state filter, and the biofilter is sprayed with a microbial culture provided from a microbial supply tank. The system is built in such a way that the microbial community formed on the surface of the biofilter can be involved in the process of treating wastewater exposed to the biofilter. Wastewater that has passed through biofilter is evaluated for removal of pollutant before discharge, and wastewater from which pollutant has been removed is discharged from the facility. (See pages 3-4, lines 129–140).

 

 

Q4. - The English must be revised. Example: line 134: "Each sample was collected at 1 L per site"

 

Response: We thank the reviewer for pointing out these errors, and we have modified the text as follows (See page 4, lines 145–146): The 1 L bottle samples were collected at each site.

 

 

Author Response File: Author Response.docx