Screening and Identification of SOB and Its Effect on the Reduction in H₂S in Dairy Farms

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

The article examines the issue of cultivating new strains of sulfate-reducing bacteria that reduce the concentration of hydrogen sulfide in gases emitted by wastewater treatment plants at livestock farms. The topic of the study is relevant due to the negative impact of these gases on human health and the environment as a whole. In this context, the bacterial strains indicated in the manuscript have not been previously considered, the cultivation method and laboratory analysis results are presented logically and in sufficient volume. However, I consider it necessary to expand the literature review on the topic of the study and indicate more relevant sources. In this case, it is necessary to supplement the description of the conditions for introducing the studied strains into real treatment facilities and the effectiveness of their impact.

 

Please comment on the following questions:

1. The inscriptions on many diagrams are difficult to read.
2. The list of references should be supplemented with a source with a shelf life of no more than 5 years.
3. Does changing pH affect the efficiency of hydrogen sulfide removal under real conditions?
4. To increase the level of information content of the literature review, it is recommended to include the source [https://doi.org/10.1016/j.jclepro.2022.134109]
5. Describe the conditions of biological wastewater treatment under which the studied SOB strains are introduced
6. Provide the equations of oxidation-reduction reactions of hydrogen sulfide formation in wastewater when introducing the SOB strain

Author Response

Comment and Suggestions 1: The inscriptions on many diagrams are difficult to read.

Response to comment 1:

Thank you very much for this comment. We have enlarged the size of Figure 1-7 to make the content clearer. In addition, we have added the identity shown in Figure 2 (a-c). We hope you can agree with our modification.

 

Comment and Suggestions 2: The list of references should be supplemented with a source with a shelf life of no more than 5 years.

Response to comment 2:

Thank you for this suggestion. According to your suggestion, we have added several references from recent years.

 

Comment and Suggestions 3: Does changing pH affect the efficiency of hydrogen sulfide removal under real conditions?

Response to comment 3:

Thank you very much for this comment. Thank you for your suggestion. From the results of our simulation experiments, changing the pH affects the removal of hydrogen sulfide (see Figure 5). However, the sulfur-oxidising bacterium AEB2, which was screened in this study, is more suitable for sulfur oxidation under neutral alkaline conditions at pH 7-10, while sulfur oxidation is not obvious under acidic conditions at pH less than 6 (as shown in the results of Figure 2). In livestock manure treatment, wastewater storage ponds (especially biogas slurry storage ponds) are one of the main sources of hydrogen sulphide emissions. The pH of the effluent from wastewater storage ponds is generally 7.0-8.5, which belongs to the suitable range for the sulfur oxidising bacteria AEB2 screened in this study to perform hydrogen sulfide removal. In the next step, we will also investigate the effect of environmental conditions such as pH and temperature on hydrogen sulfide removal under actual conditions, in order to comprehensively evaluate the effect of sulfur-oxidising bacterial strain AEB2 on the removal of hydrogen sulfide malodorous gases.

 

Comment and Suggestions 4: To increase the level of information content of the literature review, it is recommended to include the source [https://doi.org/10.1016/j.jclepro.2022.134109]

Response to comment 4:

Thank you very much for this suggestion. We have cited this reference in the Introduction.

 

Comment and Suggestions 5: Describe the conditions of biological wastewater treatment under which the studied SOB strains are introduced

Response to comment 5:

Thank you for this suggestion. The wastewater used in the experiment was obtained from the storage tank of dairy farm biogas slurry after mesophilic biogas fermentation.

 

Comment and Suggestions 6: Provide the equations of oxidation-reduction reactions of hydrogen sulfide formation in wastewater when introducing the SOB strain

Response to comment 6:

Thank you very much for this comment. According to your suggestion, the REDOX reaction equation of selected sulfur-oxidizing bacteria AEB2 is given in the manuscript.

Author Response File: Author Response.pdf

Reviewer 2 Report

Comments and Suggestions for Authors

The manuscript titled “Screening and identification of SOB and its effect on the reduction of H₂S in dairy farm” detects a sulfur-oxidizing bacteria (SOB) strain through different level’s screening. The optimal operating conditions were seeding rate 4%, temperature 30 ℃, stirring speed 90 rpm, and pH=8. Some points should be considered before final decision:

1. The Abstract section should include (a) purpose (giving the main purposes and research question), (b) methods, (c) results and discussion, and (d) conclusions (+ recommendations)
2. Don’t repeat the words of the Title in the “keywords”
3. The y-axis of Fig. 2(b) can be a yield, such as µg/L or µg/g
4. What are the suggested H₂S removal mechanisms
5. The captions, such as “Figure 2. The identification of AEB2 strain” should be written in details
6. For “Table 1. Genomic comparison between strain AEB2 and typical strains of the closely phylogenetic Halomonas genus”, you can add another column for reference
7. The “Conclusion” section should not include well know information, such as “The oxidation products of S2- were mainly elemental sulfur and tetrathionate.”
8. Add declaration statements, competing interest, acknowledgement, funding, etc.
9. The word “dairy” in the title should be comprehensively addressed throughout the manuscript.
10. What are the defense mechanisms introduced by the Halomonas sp. to withstand harsh odor conditions
11. What are the proposed management strategies for the disposal of sludge containing sulfur-oxidizing bacteria
12. Why have the authors selected H₂S; what’s the novelty here?
13. There is no deep discussion on the degradation pathway; improve this justification

Comments on the Quality of English Language

The English could be improved

Author Response

Comment and Suggestions 1: The Abstract section should include (a) purpose (giving the main purposes and research question), (b) methods, (c) results and discussion, and (d) conclusions (+ recommendations)

Response to comment 1:

Thank you very much for this suggestion. Following your suggestions, we have revised the Abstract to make it more consistent with these four sections.

(a) purpose (giving the main purposes and research question)

The problem of foul odor caused by H₂S in livestock farms has become one of the main complaints. In this study, an optimal sulfur-oxidizing bacteria (SOB) strains were screened from the dairy farm wastewater and the adjacent soil for the odor treatment of dairy farm wastewater.

(b) methods

The strains and physiological functions were determined by identification and genome comparison, and the optimal operating conditions were determined by experiments under different conditions.

(c) results and discussion

The identification results showed that the strain had the highest homology with Halomonas mongoliensis and named Halomonas sp. AEB2. The comparative genomic results showed that the average nucleotide identity and DNA-DNA hybridization value were 95.8% and 68.6%, respectively. The optimization results were: sodium succinate-carbon (10 g/L), ammonium chloride-nitrogen (0.07 g/L). The optimal operating conditions were as follows: seeding rate 4%, temperature 30 ℃, stirring speed 90 rpm and pH=8. The oxidation products of AEB2 were mainly elemental sulfur and tetrathionate; and the metabolic pathway of AEB2 was constructed accordingly.

(d) conclusions (+ recommendations)

The study suggests a feasible path to reduce H₂S emissions from dairy farm,and will provide theoretical support for the restoration of livestock environment and sustainability.

 

Comment and Suggestions 2: Don’t repeat the words of the Title in the “keywords”

Response to comment 2:

Thank you very much for this comment. We have modified the keywords, and the modified keywords are not repeated with the Title. Moreover, we have added relevance to journals in abstracts and keywords as follows:

Abstract: The problem of foul odor caused by H₂S in livestock farms has become one of the main complaints. In this study, an optimal sulfur-oxidizing bacteria (SOB) strains were screened from the dairy farm wastewater and the adjacent soil for the odor treatment of dairy farm wastewater. The strains and physiological functions were determined by identification and genome comparison, and the optimal operating conditions were determined by experiments under different conditions. The identification results showed that the strain had the highest homology with Halomonas mongoliensis and named Halomonas sp. AEB2. The comparative genomic results showed that the average nucleotide identity and DNA-DNA hybridization value were 95.8% and 68.6%, respectively. The optimization results were: sodium succinate-carbon (10 g/L), ammonium chloride-nitrogen (0.07 g/L). The optimal operating conditions were as follows: seeding rate 4%, temperature 30 ℃, stirring speed 90 rpm and pH=8. The oxidation products of AEB2 were mainly elemental sulfur and tetrathionate; and the metabolic pathway of AEB2 was constructed accordingly. The study suggests a feasible path to reduce H₂S emissions from dairy farm,and will provide theoretical support for the restoration of livestock environment and sustainability.

Keywords: Sulfur-oxidizing bacteria; Biological decdorization; Halomonas; Heterotrophic oxidation; Oxidation pathway; Sustainability

 

Comment and Suggestions 3: The y-axis of Fig. 2(b) can be a yield, such as µg/L or µg/g

Response to comment 3:

Thank you for this comment. We have modified the units of y-axis in Figure 1b (Figure 2 is strain identification, does not involve x-axis and y-axis, we presume you should be referring to Figure 1).

 

Comment and Suggestions 4: What are the suggested H₂S removal mechanisms

Response to comment 4:

Thank you very much for this suggestion. The mechanism of hydrogen sulphide removal by the sulphur-oxidising bacterium AEB2 relies mainly on the two genes it carries, sqr and fccA/B, which oxidise hydrogen sulphide to monomeric sulphur or sodium thiosulphate.

 

Comment and Suggestions 5: The captions, such as “Figure 2. The identification of AEB2 strain” should be written in details

Response to comment 5:

Thank you very much for this comment. We have changed the caption for Figure 2 (the detailed name for Figure 2a-c) to make its content more detailed.

 

Comment and Suggestions 6: For “Table 1. Genomic comparison between strain AEB2 and typical strains of the closely phylogenetic Halomonas genus”, you can add another column for reference

Response to comment 6:

Thank you for this comment. According to your suggestion, we added a line of AEB2 in Table 1 as a comparison.

 

Comment and Suggestions 7: The “Conclusion” section should not include well know information, such as “The oxidation products of S^2- were mainly elemental sulfur and tetrathionate.”

Response to comment 7:

Thank you very much for this suggestion. We have deleted the well known information from the Conclusion.

 

Comment and Suggestions 8: Add declaration statements, competing interest, acknowledgement, funding, etc.

Response to comment 8:

Thank you very much for this comment. We added fundings and conflicts of interest at the end of the manuscript (before references). We do not involve acknowledgements in this manuscript. We hope you can agree with our modification.

 

Comment and Suggestions 9: The word “dairy” in the title should be comprehensively addressed throughout the manuscript.

Response to comment 9:

Thank you very much for this comment. We have modified all the words about "dairy" in the manuscript, it should be "dairy farm wastewater".

 

Comment and Suggestions 10: What are the defense mechanisms introduced by the Halomonas sp. to withstand harsh odor conditions

Response to comment 10:

Thank you for this comment. Hydrogen sulphide malodorous gas is toxic to microorganisms and affects bacterial reproduction and metabolism. In this paper, sulfur-oxidising bacterium AEB2 was isolated from a dairy farm biogas slurry storage tank with a large production of hydrogen sulfide and other malodorous gases, and the strain has a good adaptability to this harsh condition. At the same time, strain AEB2 has a highly efficient hydrogen sulphide oxidation (see Figure 6 and Table 2), which can oxidise hydrogen sulphide to monosodium sulphur, which is essentially non-toxic to bacteria, in a relatively short period of time (4 h). This may be the mechanism by which the sulfur-oxidising bacterium AEB2 resists malodorous gases. The detailed mechanism of action needs to be further investigated.

 

Comment and Suggestions 11: What are the proposed management strategies for the disposal of sludge containing sulfur-oxidizing bacteria

Response to comment 11:

Thank you very much for this suggestion. For the wastewater inoculated with sulfur oxidizing bacteria AEB2, in order to better play the role of hydrogen sulfide removal, the temperature of the sewage can be kept above 15℃ in management, and the pH can be controlled above 7.

 

Comment and Suggestions 12: Why have the authors selected H₂S; what’s the novelty here?

Response to comment 12:

Thank you very much for this suggestion. It was found that H₂S is the main malodorous gas emitted during livestock manure storage, accounting for more than 65% of the total. Therefore, the removal of H₂S is of great significance for the effective reduction and control of malodour gases in livestock farms. Currently, there are several strains that exert hydrogen sulfide removal effect in alkaline, high sodium ion livestock and poultry digestate environment, which is the novelty of this study.

 

Comment and Suggestions 13: There is no deep discussion on the degradation pathway; improve this justification

Response to comment 13:

Thank you very much for this comment. In this research, the authors have analysed and discussed the metabolic pathway of hydrogen sulfide in the context of the oxidation products of hydrogen sulfide by the sulfur-oxidising bacterium AEB2 as well as the whole genome sequence of the bacterium, and basically determined the oxidation pathway of hydrogen sulfide by AEB2. However, more methods are needed to analyse the sulfur metabolic pathway of the bacteria in depth. If necessary, we will carry out the detailed sulfur metabolic pathway analysis of sulfur-oxidising strain AEB2 in combination with stable isotope probes and other methods in the next step.

Author Response File: Author Response.pdf

Reviewer 3 Report

Comments and Suggestions for Authors

1.Consider adding more specific terms such as “biological deodorization”, “Halomonas”, “heterotrophic oxidation”.

2.With unclear articulation of knowledge gaps. Focus on key problem and condense text.

3.Only one-way ANOVA was applied. Recommend inclusion of multifactorial ANOVA or regression significance tests.

4.In oxidation tests, include sterilized control, SOB-free control, etc., to enhance experimental rigor.

5.Many phrases are unclear, such as “subunit of sulfide dehydrogenase”. Careful language polishing is required.

6.ANI is borderline and DDH is insufficient (<70%). More taxonomic evidence is needed before suggesting a novel species.

7.No evaluation of stability, scalability, or cost of SOB application in real-world settings.

8.Should emphasize application potential, implementation suggestions, and study limitations.

Comments on the Quality of English Language

The English could be improved to more clearly express the research.

Author Response

Comment and Suggestions 1: Consider adding more specific terms such as “biological deodorization”, “Halomonas”, “heterotrophic oxidation”.

Response to comment 1:

Thank you very much for this suggestion. The keywords in the manuscript are modified. The modified keywords are “Sulfur-oxidizing bacteria; Biological decdorization; Halomonas; Heterotrophic oxidation; Oxidation pathway; Sustainability”.

 

Comment and Suggestions 2: With unclear articulation of knowledge gaps. Focus on key problem and condense text.

Response to comment 2:

Thank you very much for this comment. According to your suggestion, it has been modified in the manuscript.

 

Comment and Suggestions 3: Only one-way ANOVA was applied. Recommend inclusion of multifactorial ANOVA or regression significance tests.

Response to comment 3:

Thank you for this comment. As you suggested, the significance test was added (Figure 1b and Figure 5).

 

Comment and Suggestions 4: In oxidation tests, include sterilized control, SOB-free control, etc., to enhance experimental rigor.

Response to comment 4:

Thank you very much for this comment. In the oxidation tests, for the rigor of the test, the authors set controls with uninoculated SOB (see Figure 1a and Figure 6).

 

Comment and Suggestions 5: Many phrases are unclear, such as “subunit of sulfide dehydrogenase”. Careful language polishing is required.

Response to comment 5:

Thank you very much for this suggestion. Changes have been made to the text as you suggested.

 

Comment and Suggestions 6: ANI is borderline and DDH is insufficient (<70%). More taxonomic evidence is needed before suggesting a novel species.

Response to comment 6:

Thank you very much for this suggestion. Indeed, in addition to ANI and dDDH analyses based on the whole genome of bacteria, the identification of new species also requires the determination of taxonomic indicators such as fatty acids, respiratory quinones, and polar lipids for comparative confirmation. In this research, the taxonomic status of isolated sulfur-oxidizing bacteria AEB2 was only speculated based on the results of ANI and DDH. Further identification results will be carried out in the next study.

 

Comment and Suggestions 7: No evaluation of stability, scalability, or cost of SOB application in real-world settings.

Response to comment 7:

Thank you very much for this comment. The evaluation of the stability, scalability, and cost of SOB in the application needs to be carried out in the actual environment after continuous and extensive application tests. The focus of this research is to screen efficient SOB strains, verify their sulfur oxidation performance, and clarify their hydrogen sulfide removal mechanism, so as to provide a basis for the next step in the actual environment application. In the next step, we will carry out the reduction effect of hydrogen sulfide bacteria on hydrogen sulfide odor gas under different actual environmental conditions, such as inoculation amount, sewage concentration, season, etc. On this basis, the application stability and application cost of the strain were evaluated.

 

Comment and Suggestions 8: Should emphasize application potential, implementation suggestions, and study limitations.

Response to comment 8:

Thank you for this comment. We have added this section to the Conclusion as follows:

In order to reduce the emission of H₂S from livestock farms, the strains were screened from dairy farm wastewater. The nutrient composition and operating parameters were optimized, and the oxidation intermediates were analyzed. The possible metabolic pathways were revealed by whole genome sequencing. The results showed that the strain had a high H₂S removal rate of 47.1% in dairy farm wastewater. The strain had a wide range of pH and temperature applications. The oxidation products of S^2- are mainly elemental sulfur and sodium thiosulfate. The screened halomonas AEB2 showed a good sulfur oxidation effect, and had a good application prospect in reducing the odor gas of hydrogen sulfide in livestock wastewater. The detailed sulfur metabolism mechanism of the strain and its stability and cost in the actual environment need further study.

 

Author Response File: Author Response.pdf

Round 2

Reviewer 2 Report

Comments and Suggestions for Authors

The authors responses are satisfactory

Reviewer 3 Report

Comments and Suggestions for Authors

Accept