Review Reports - <i>Anaerococcoides</i> <i>asporogena</i> gen. nov., sp. nov., a Strictly Anaerobic Bacterium, Isolated from the Dehydrated Sludge of a Steel Factory’s Wastewater Treatment Plant

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

This study reports the isolation and characterization of strain QWL-01 from sewage sludge of a steel factory wastewater treatment plant. The strain is a strictly anaerobic, non-spore-forming, coccoid bacterium with distinct physiological and chemotaxonomic features. Phylogenetic and genomic analyses, including 16S rRNA similarity, ANI, AAI, and dDDH values, demonstrate that it represents a novel lineage within the family Clostridiaceae. Based on combined phenotypic and genomic evidence, the authors propose a new genus and species, Anaerococcoides asporogenes gen. nov., sp. nov. However, there are a few points to note in the manuscript.

The author needs to cite the genus delineation thresholds (AAI: 60–80%; 16S: ~94.5%) in the introduction. In addition, it is recommended that authors add a clear taxonomic justification paragraph, such as why not a new species within Proteiniclasticum or Youngiibacter? Highlight distinct phenotypic and chemotaxonomic traits more clearly.
The manuscript is descriptive but lacks metabolic reconstruction and ecological role in sludge. If possible, please add fermentation pathways from the genome, a discussion of substrate utilization, and a Possible role in carbon cycling and anaerobic digestion.
In Figure 2, the description is minimal; it is suggested that the tree construction details, a substitution model, an alignment method, and a Bayesian or NJ tree be added
In the Methods section, the DSM 120 medium composition is repeated, and NH4Cl appears twice. Remove duplication. Also, “uncultured microorganisms” is mentioned multiple times; please avoid repetition.
There are some typographical errors, for example, “cultured-dependent method” should be “culture-dependent method.” “Anaer-obic bacterium” has a formatting issue, “2.1..” Remove the double period; “bioinfomatics” should be “bioinformatics.” And “Chain” should be “China.”
Units and formatting inconsistencies, for the temperature: “℃” vs “°C” use °C consistently
There are some spacing issues, for example, “0-3% NaCl” to “0–3% (w/v) NaCl.”
The μm unit formatting is inconsistent in the manuscript; need to fix it.
In the Figure and Table, figure references are inconsistent, for example, “Fig. 1a-d” vs “Fig. 1a–d”. In Table 2 formatting, messy fatty acid entries and inconsistent symbols (ꞷ vs ω)
In References, it was observed that the DOI is incomplete. Please check and correct it. In addition, the formatting of Journal names is inconsistent. “Isme j” should be ISME J. Please check the capitalization, as it is inconsistent.

Author Response

Thank you for your detailed revisions. All revised parts have been highlighted in yellow for ease of reference.

Reviewer 1

Comments 1-1: [The author needs to cite the genus delineation thresholds (AAI: 60–80%; 16S: ~94.5%) in the introduction. In addition, it is recommended that authors add a clear taxonomic justification paragraph, such as why not a new species within Proteiniclasticum or Youngiibacter? Highlight distinct phenotypic and chemotaxonomic traits more clearly.]

Response 1-1: [We thank the reviewer for this helpful suggestion. In the revised manuscript, we have added the commonly used genus delineation thresholds in the Introduction (Line 88–97), including the approximate 16S rRNA gene threshold of 94.5% and the proposed AAI genus range of 60–80%. We have also added a clearer taxonomic justification paragraph explaining why strain QWL-01ᵀ is proposed as a novel genus rather than a novel species within Youngiibacter or Proteiniclasticum (Line 309–328). Specifically, strain QWL-01ᵀ shows 16S rRNA gene similarities of only 93.82% and 93.75% to the closest type strains of these genera, and AAI values of only 49.27–51.58%, all of which are below the commonly accepted genus-level reference ranges. In addition, we now more clearly highlight its distinguishing phenotypic and chemotaxonomic features, including its coccoid morphology and characteristic fatty acid profile (in section 4.6).]

Comments 1-2: [The manuscript is descriptive but lacks metabolic reconstruction and ecological role in sludge. If possible, please add fermentation pathways from the genome, a discussion of substrate utilization, and a Possible role in carbon cycling and anaerobic digestion.]

Response 1-2: [Thank you for your comments. Some revisions were made as follows.

Line 369: Added “Section 4.4. Predicted fermentative metabolism and ecological role of QWL-01^T” to introduce the fermentation pathways, potential substrate utilization, and the possible role of this strain in carbon cycling based on genomic data.
Line 396: Added Section 4.5, “Putative Detoxification and Pollutant Transformation Potential of Strain QWL-01ᵀ,” to describe the potential heavy metal detoxification and pollutant transformation capabilities inferred from the genome of strain QWL-01ᵀ.]

Comments 1-3: [In Figure 2, the description is minimal; it is suggested that the tree construction details, a substitution model, an alignment method, and a Bayesian or NJ tree be added.]

Response 1-3: [Thank you for your comments. Some revisions were made as follows.

On the new lines 190-194: the original sentence of “The 16S rRNA gene phylogenetic tree was reconstructed by the MEGA X program [27] using the Maximum Likelihood Method with 1000 bootstrap replicates.” was revised as “Sequence alignment was performed using the Clustal W program [34]. The 16S rRNA gene phylogenetic tree was reconstructed by the Maximum-Likelihood (ML) [35], Neighbor-Joining (NJ) [36] and Minimum-Evolution (ME) [37] algorithms, using the MEGA X program with Maximum Composite Likelihood substitution model [38].”
On the new lines 294-295: added the sentence “Sequence alignment was performed using the Clustal W program.” after “… and the related genera.”.
On the new lines 296-297: the original sentence of “Bootstrap values at nodes are the percentages of 1000 replicates.” was revised as “Bootstrap values at the nodes are shown as percentages based on an ML analysis of 1,000 resampled datasets.”
On the new line 299: added “●, ML, NJ and ME on one branch.” after “… evolutionary distances.”.
Added new references of [34-38].

Comments 1-4: [In the Methods section, the DSM 120 medium composition is repeated, and NH4Cl appears twice. Remove duplication. Also, “uncultured microorganisms” is mentioned multiple times; please avoid repetition.]

Response 1-4: [Thanks for your detailed review. Some revisions were made as follows.

On the original line 83: deleted “NH₄Cl, 1.0 g;”
On the line 113-114: revised as “Na₂S^.9H₂O, 0.3 g and 0.1% (w/v) resazurin, 0.5 mL.” after “…tryptone, 2 g;”.
We checked the occurrence of the term “uncultured microorganisms” in the manuscript and found that it appears only once. Therefore, no revision was made in response to this comment.

Comments 1-5: [There are some typographical errors, for example, “cultured-dependent method” should be “culture-dependent method.” “Anaer-obic bacterium” has a formatting issue, “2.1..” Remove the double period; “bioinfomatics” should be “bioinformatics.” And “Chain” should be “China.”]

Response 1-5: [Thank you for your detailed review. We have made the following revisions.

Line 3: replaced ‘anaer-obic’ with ‘anaerobic’.
Line 33: replaced ‘cultured-dependent method’ with ‘culture-dependent method’.
Removed double period for ‘2.1.. ~ 2.6.. and 3.1..~3.5.., 4.1..~4.3..’, respectively.
Line 218: replaced ‘bioinfomatics’ with ‘bioinformatics’.
Line 224: replaced ‘Chain’ with ‘China’.]

Comments 1-6: [Units and formatting inconsistencies, for the temperature: “℃” vs “°C” use °C consistently]

Response 1-6: [Thank you for your comments. All the temperature signs of ‘℃’ were replaced with ‘^oC’.]

Comments 1-7: [There are some spacing issues, for example, “0-3% NaCl” to “0–3% (w/v) NaCl.”]

Response 1-7: [Thanks for your comments. All “hyphen” between two numbers were replaced with “dash”.

Line 113, 163, 262: added “(w/v)” before “NaCl”.
Line 146, 232: added “(w/v)” before “KOH”.
Line 242: added “(w/v)” before “uranyl acetate”.

Comments 1-8: [The μm unit formatting is inconsistent in the manuscript; need to fix it.]

Response 8: [Thank you for your comment. We have revised the manuscript to ensure consistent formatting of the unit “μm” throughout, including spacing and range presentation.]

Comments 1-9: [In the Figure and Table, figure references are inconsistent, for example, “Fig. 1a-d” vs “Fig. 1a–d”. In Table 2 formatting, messy fatty acid entries and inconsistent symbols (ꞷ vs ω)]

Response 1-9: [Thank you for your comments. Some revisions were made as follows.

Line 228-229: replaced the both “hyphen” with “dash”.
In Table 2 and line 269: replaced “ꞷ” with “ω”.
In Table 2: divided the original lane labeled “16:1 ω7c or 16:1 ω6c” into two separate lanes, “16:1 ω6c” and “16:1 ω7c”.

Comments 1-10: [In References, it was observed that the DOI is incomplete. Please check and correct it. In addition, the formatting of Journal names is inconsistent. “Isme j” should be ISME J. Please check the capitalization, as it is inconsistent.]

Response 1-10: [Thanks for your comments. The format of references has been revised.]

Reviewer 2 Report

Comments and Suggestions for Authors

The manuscript presents the taxonomic characterization of a novel anaerobic bacterium isolated from sludge, and overall the study follows a standard polyphasic approach. The genomic data, especially low 16S rRNA similarity and ANI/AAI values, support the proposal of a new genus. However, the manuscript in its current form lacks depth in phylogenomic justification, chemotaxonomic completeness, and ecological interpretation.

The authors must strengthen the justification for proposing a novel genus beyond sequence similarity thresholds. While 16S rRNA similarity below 95% is generally considered supportive of genus delineation, current taxonomic standards increasingly rely on genome-based phylogeny (GTDB, phylogenomic frameworks). The manuscript relies heavily on 16S-based phylogeny (Fig. 2), which is insufficient alone. The authors should include a robust genome-based phylogenomic tree using conserved marker genes (e.g., GTDB-Tk or PhyloPhlAn), and explicitly compare their results with accepted genus boundaries (AAI ~60–65%). This will significantly strengthen the taxonomic claim.
The phylogenetic tree needs improvement in taxon selection and resolution. The current tree does not clearly show proper outgroup usage or strong bootstrap support. The authors should include more closely related taxa and reconstruct the tree using a more reliable method.
The ANI, dDDH, and AAI values are reported but not properly interpreted. The authors should clearly state accepted genus-level thresholds and explain how their strain fits within these criteria. This is essential to support the taxonomic proposal.
The contradiction between Gram staining (Gram-negative) and genomic inference (Gram-positive type) must be clearly explained with literature support. This is a known phenomenon in some Firmicutes, but it should be properly discussed.
The chemotaxonomic characterization is incomplete. Key features such as polar lipids, quinones, and peptidoglycan type are missing. These are important for genus-level description and should be included or justified.
The Biolog AN results appear very limited (only three substrates positive). The authors should justify the method under strict anaerobic conditions and compare with related genera to show meaningful differences.
The ecological relevance is weak. The introduction discusses wastewater microbiology, but the discussion does not connect the isolate to its ecological role. The authors should elaborate on possible functions in sludge systems.
The physiological comparison (Table 1) needs better interpretation. The authors should clearly highlight which traits are truly diagnostic, rather than listing minor differences.
The genome analysis lacks functional insight. The authors should discuss key metabolic pathways or functional genes to provide biological relevance.
The isolation strategy is not clearly described. The term “culturomic approach” is used, but the methodology should be explained in more detail.
The manuscript lacks comparison with recent studies in Clostridiaceae. The authors should include recent literature and position their work within current taxonomy trends.
The supplementary data are not well integrated. Important results from ANI/dDDH tables should be discussed in the main text.

Author Response

Thank you for your detailed revisions. All revised parts have been highlighted in yellow for ease of reference.

Reviewer 2

Comments 2-1: [The authors must strengthen the justification for proposing a novel genus beyond sequence similarity thresholds. While 16S rRNA similarity below 95% is generally considered supportive of genus delineation, current taxonomic standards increasingly rely on genome-based phylogeny (GTDB, phylogenomic frameworks). The manuscript relies heavily on 16S-based phylogeny (Fig. 2), which is insufficient alone. The authors should include a robust genome-based phylogenomic tree using conserved marker genes (e.g., GTDB-Tk or PhyloPhlAn), and explicitly compare their results with accepted genus boundaries (AAI ~60–65%). This will significantly strengthen the taxonomic claim.]

Responses 2-1: [We appreciate the reviewer’s suggestion. In the original manuscript, we have a genome-based phylogenetic analysis using TYGS, which generates a genome-scale GBDP tree and type-strain-based genomic comparisons. The TYGS analysis supported the placement of strain QWL-01ᵀ as a distinct lineage separate from the genera Youngiibacter and Proteiniclasticum (Fig. S5). Although TYGS uses a genome distance-based framework rather than a conserved marker gene set, the resulting phylogenetic placement was consistent with both the 16S rRNA gene phylogeny and the AAI results. In addition, we revised the text in Lines 309-328 to explicitly note that the AAI values of strain QWL-01ᵀ are below the suggested genus-level boundary, which further supports its assignment to a novel genus.]

Comments 2-2: [The phylogenetic tree needs improvement in taxon selection and resolution. The current tree does not clearly show proper outgroup usage or strong bootstrap support. The authors should include more closely related taxa and reconstruct the tree using a more reliable method.]

Responses 2-2: [We thank the reviewer for this helpful suggestion. We agree that the original 16S rRNA gene phylogenetic tree required improvement. In the revised manuscript, we reconstructed the 16S rRNA gene tree using the Maximum-Likelihood (ML), Neighbor-Joining (NJ), and Minimum-Evolution (ME) methods. We also revised the text in Lines 190–194 and the figure legend to clearly describe the phylogenetic methods used. The revised tree provides improved resolution and shows that strain QWL-01ᵀ forms a distinct lineage separate from Youngiibacter and Proteiniclasticum. This 16S rRNA gene phylogeny is also consistent with the genome-based evidence obtained from TYGS analysis and AAI comparisons.]

Comments 2-3: [The ANI, dDDH, and AAI values are reported but not properly interpreted. The authors should clearly state accepted genus-level thresholds and explain how their strain fits within these criteria. This is essential to support the taxonomic proposal.]

Responses 2-3: [We thank the reviewer for this important comment. In the revised manuscript, we have added the commonly accepted taxonomic reference values for species- and genus-level delineation based on 16S rRNA gene sequence similarity, ANI, dDDH, and AAI in Lines 88–97. We also revised the text in Lines 309-328 to more clearly explain how strain QWL-01ᵀ fits within these criteria and how these results support its assignment to a novel genus.]

Comments 2-4: [The contradiction between Gram staining (Gram-negative) and genomic inference (Gram-positive type) must be clearly explained with literature support. This is a known phenomenon in some Firmicutes, but it should be properly discussed.]

Responses 2-4: [We thank the reviewer for this important comment. We agree that the apparent discrepancy between the Gram-staining result and the genomic inference should be clarified. In the revised manuscript, we revised the text of “section 4.1” to discuss that Gram-stain reaction does not always directly reflect phylogenetic affiliation or the underlying cell-envelope architecture. Although strain QWL-01ᵀ was observed to stain Gram-negative, its phylogenetic position and genomic features support its affiliation with the family Clostridiaceae, a lineage generally characterized by a Gram-positive-type cell envelope. We further note that this phenomenon has been reported in several members of Firmicutes/Bacillota, particularly among clostridial lineages, in which cells may appear Gram-negative or Gram-variable despite belonging to taxa with Gram-positive-type cell wall organization. Other revisions were made as follows.

On the line 35: replaced “Gram-positive” with “Gram-stain-negative”.
On the lines 232-234: replaced “…3% (w/v) KOH, supporting the presence of a monoderm-type cell envelope and suggesting that the negative Gram-staining result reflects atypical cell wall properties rather than a true diderm structure” with “…3% (w/v) KOH, supporting a monoderm, Gram-positive-type cell envelope organization and suggesting that the Gram-stain-negative result reflects atypical cell wall properties rather than a true diderm structure”
On the line 464: added “Gram-stain-negative” after “…, non-motile,”]

Comments 2-5: [The chemotaxonomic characterization is incomplete. Key features such as polar lipids, quinones, and peptidoglycan type are missing. These are important for genus-level description and should be included or justified.]

Responses 2-5: [We thank the reviewer for this important comment. We agree that polar lipids, quinones, and peptidoglycan type are valuable chemotaxonomic traits for genus-level description in traditional polyphasic taxonomy. Nevertheless, the placement of strain QWL-01ᵀ is strongly supported by multiple independent lines of evidence, including 16S rRNA gene phylogeny, TYGS-based genome phylogeny, ANI, dDDH, AAI, phenotypic characteristics, and cellular fatty acid composition. Recent taxonomic recommendations also suggest that, in the genome era, routine chemotaxonomic testing should not necessarily be considered mandatory when genome-based evidence is sufficient, although such data remain informative and desirable (Vandamme and Sutcliffe, 2021; Riesco and Trujillo, 2024). ]

Comments 2-6: [The Biolog AN results appear very limited (only three substrates positive). The authors should justify the method under strict anaerobic conditions and compare with related genera to show meaningful differences.]

Responses 2-6: [We thank the reviewer for this helpful comment. We agree that the Biolog AN results of strain QWL-01ᵀ were limited and should be interpreted cautiously. In the revised manuscript, we have clarified the testing conditions and the taxonomic significance of these data. Some revisions were made as follows.

On the lines 173-175: added the sentence of “The Biolog AN inoculum preparation and incubation were carried out using pre-reduced medium and strict anaerobic handling to minimize oxygen exposure” to clarify the testing condition.
On the lines 410-418: added a paragraph “The Biolog AN results for strain QWL-01ᵀ were limited……, supporting its phenotypic distinctiveness in conjunction with the phylogenetic and genomic evidence.]

Comments 2-7: [The ecological relevance is weak. The introduction discusses wastewater microbiology, but the discussion does not connect the isolate to its ecological role. The authors should elaborate on possible functions in sludge systems.]

Responses 2-7: [Thank you for your comments. Some revisions were made as follows.

On line 369: Added a paragraph of section “4.4. Predicted fermentative metabolism and ecological role of QWL-01^T” to introduce the fermentation pathways, potential substrate utilization, and the possible role of this strain in carbon cycling based on genomic data.
On line 396: Added a paragraph of section “4.5. Putative detoxification and pollutant transformation potential of strain QWL-01ᵀ” to describe the potential of strain QWL-01ᵀ for detoxification and pollutant transformation.

Comments 2-8: [The physiological comparison (Table 1) needs better interpretation. The authors should clearly highlight which traits are truly diagnostic, rather than listing minor differences.]

Responses 2-8: [Thank you for this helpful comment. We agree that the physiological comparison should emphasize the traits with the greatest diagnostic value. First, cell morphology represents a major distinguishing feature between strain QWL-01ᵀ and the genera Youngiibacter and Proteiniclasticum. As highlighted in Lines 234–236, within these two genera, only Proteiniclasticum aestuarii JCM 34531ᵀ exhibits a coccoid morphology, whereas all other species are rod-shaped (Table 1). Second, the genomic G+C content of strain QWL-01ᵀ differs notably from those of both Youngiibacter and Proteiniclasticum. As stated in Lines 302-305, the G+C content of strain QWL-01ᵀ differs from those of the two Youngiibacter type strains (44.84–46.57%) and the three Proteiniclasticum type strains (43.07–51.35%), with differences of 4.24–5.97% and 0.54–7.73%, respectively (Tables 1 and S3).]

Comments 2-9: [The genome analysis lacks functional insight. The authors should discuss key metabolic pathways or functional genes to provide biological relevance.]

Responses 2-9: [Thank you for your comments. In the revised manuscript, we added two paragraphs in Sections 4.4 and 4.5 to discuss key metabolic pathways and functional genes, thereby providing greater biological relevance.]

Comments 2-10: [The isolation strategy is not clearly described. The term “culturomic approach” is used, but the methodology should be explained in more detail.]

Response 2-10: [Thank you for your comments. We did employ a small-scale culturomics approach, using 30 methanogen media under four different cultivation conditions, to isolate anaerobes from sewage sludge. Strain QWL-01ᵀ was recovered from modified DSM 120 medium incubated at room temperature. Therefore, in Section 2.2, we described only the specific isolation conditions for strain QWL-01ᵀ. To clarify the overall cultivation strategy, we have added a description of the small-scale culturomics approach in the Introduction (Lines 128–139).]

Comments 2-11: [The manuscript lacks comparison with recent studies in Clostridiaceae. The authors should include recent literature and position their work within current taxonomy trends.]

Responses 2-11: [Thank you for your comments. In the revised manuscript, we added a paragraph in Section 4.7 to discuss recent taxonomic studies on Clostridiaceae and related anaerobic lineages, and to better position our work within current genome-based taxonomic trends.]

Comments 2-12: [The supplementary data are not well integrated. Important results from ANI/dDDH tables should be discussed in the main text.]

Responses 2-12: [We thank the reviewer for this helpful comment. We agree that the ANI/dDDH-related results should be more fully integrated into the main text. In the revised manuscript, we have added the key ANI, dDDH, and AAI values to the main text and clarified how these values compare with the accepted reference thresholds for species- and genus-level delineation (Lines 309-328).]

Reviewer 3 Report

Comments and Suggestions for Authors

The manuscript entitled " Anaerococcoides asporogenes gen. nov., sp. nov., a strictly anaerobic bacterium, isolated from dehydrated sludge of a steel factory’s waste water treatment plant" is addresses microbial community study of dehydrated sludge collected from a steel factory’s wastewater treatment plant. Based on phenotypic, physiological, phylogenetic, and genomic relatedness evidence, strain QWL-01T represents a novel genus in the family Clostridiaceae, for which the name Anaerococcoides asporogenes gen. nov. sp. nov. is proposed. This manuscript aligns well with the aims and scope of Microorganisms. I am ready to recommend it for publication, subject to the following comments:

The introduction is too short; it should be expanded by 2-3 times. It is important to discuss the small-scale culturomic approach in more detail as a progressive new approach.
The abstract, and perhaps even the title, should also mention the small-scale culturomic approach.
The materials and methods should begin with sampling, detailing how it was carried out, how many samples were collected, and their relevance. The reader would also be interested in learning about the physicochemical conditions of the sampling site (temperature, pH, sampling depth, oxygen availability at the sampling point, etc.).
3.1. should be moved to the materials and methods.
Was the entire community analyzed using 16S rRNA gene analysis? It would be interesting to consider the contribution of the isolated strain to the community; is it dominant or a minor species?
I propose combining the results and discussion into one section; the results are presented in the discussion section.
The materials and methods provide information about a whole-genome analysis of the strain, but the text contains virtually no information about its metabolic capabilities.
The conclusion section is very brief; it should be supplemented with data on the possible role of this strain in the community, as well as a discussion of its biotechnological potential. This would significantly strengthen the manuscript.

Author Response

Thank you for your detailed revisions. All revised parts have been highlighted in yellow for ease of reference.

Reviewer 3

Comments 3-1: [The introduction is too short; it should be expanded by 2-3 times. It is important to discuss the small-scale culturomic approach in more detail as a progressive new approach.]

Responses 3-1: [Thank you for your comments. In response to the reviewers’ suggestions, we expanded the Introduction in the revised manuscript by adding three paragraphs that describe the genome-based taxonomic framework and summarize the results of our small-scale culturomics approach (Lines 74-97).]

Comments 3-2: [The abstract, and perhaps even the title, should also mention the small-scale culturomic approach.]

Responses 3-2: [Thank you for your comments. In this study, we did apply a small-scale culturomics approach, using 30 methanogen media under four different cultivation conditions, to isolate anaerobic bacteria from sewage sludge. Strain QWL-01ᵀ was specifically recovered from modified DSM 120 medium incubated at room temperature, and for this reason Section 2.2 describes only the isolation conditions directly relevant to this strain. To better explain the overall cultivation strategy, we have added a description of the small-scale culturomics approach in the Introduction (Lines 74–87). We also appreciate the suggestion regarding the title; however, we prefer to retain the current title without explicitly referring to the small-scale culturomics approach.]

Comments 3-3: [The materials and methods should begin with sampling, detailing how it was carried out, how many samples were collected, and their relevance. The reader would also be interested in learning about the physicochemical conditions of the sampling site (temperature, pH, sampling depth, oxygen availability at the sampling point, etc.).]

Responses 3-3: [Thank you for your comments. During sampling, approximately 2 kg of dehydrated sewage sludge was collected from the sludge dryer (Fig. S1) into a plastic bag. However, we apologize that no further environmental information, such as temperature, pH, or oxygen availability at the sampling point, was recorded. The relevant text has been revised accordingly as follows.

On line 128-131: the original sentence of “The sample of sludge was inoculated into the anaerobic modified DSM 120 medium without the addition of acetate and methanol, prepared according to the instruction of the medium, and incubated at room temperature (~25 °C) for 2 weeks” was revised as “Approximately 2 kg of sludge was transported to the laboratory in a plastic bag within 2 h of sampling, and about 3 mL of sludge, collected using a syringe, was inoculated into anaerobic modified DSM 120 medium and incubated at room temperature (~25 °C) for 2 weeks.”]

Comments 3-4: [3.1. should be moved to the materials and methods.]

Responses 3-4: [Thank you for your comment. We understand the reviewer’s suggestion; however, we consider the deposition numbers assigned by the different culture collection centers to be part of the outcome of the isolation and characterization of strain QWL-01ᵀ. Therefore, we have retained the original Section 3.1 in the Results section.].

Comments 3-5: [Was the entire community analyzed using 16S rRNA gene analysis? It would be interesting to consider the contribution of the isolated strain to the community; is it dominant or a minor species?]

Responses 3-5: [Thank you for this valuable comment. We agree that relating strain QWL-01ᵀ to the original sludge community would provide useful ecological context. Although 16S rRNA gene amplicon sequencing of the sludge microbiota was conducted as part of our broader study, these results were not included in the present manuscript because our primary aim here was to report the isolation and taxonomic characterization of strain QWL-01ᵀ.]

Comments 3-6: [I propose combining the results and discussion into one section; the results are presented in the discussion section.]

Responses 3-6: [Thank you for your suggestion. We understand the reviewer’s point; however, we prefer to retain separate Results and Discussion sections in this manuscript because a clear distinction between the observed data and their taxonomic interpretation is important for the description of a novel taxon. We have nevertheless revised the Discussion to improve its focus and to reduce overlap with the Results section.]

Comments 3-7: [The materials and methods provide information about a whole-genome analysis of the strain, but the text contains virtually no information about its metabolic capabilities.]

Responses 3-7: [Thank you for this helpful comment. We agree that the original manuscript focused mainly on the taxonomic use of the whole-genome data and did not sufficiently discuss the biological implications of the genome. In the revised manuscript, we have therefore added new text in Sections 4.4 and 4.5 to describe the genome-inferred metabolic pathways and functional genes of strain QWL-01ᵀ. These additions include its predicted fermentative metabolism, substrate utilization potential, and putative detoxification and pollutant transformation capabilities. Together, these revisions provide a more complete biological interpretation of the genome beyond its taxonomic application.]

Comments 3-8: [The conclusion section is very brief; it should be supplemented with data on the possible role of this strain in the community, as well as a discussion of its biotechnological potential. This would significantly strengthen the manuscript.]

Responses 3-8: [Thank you for this helpful suggestion. We agree that the original Conclusion section was too brief and did not sufficiently highlight the possible ecological and biotechnological relevance of strain QWL-01ᵀ. In the revised manuscript, we expanded the Conclusion to better summarize its potential role in the sludge community and its possible biotechnological significance. Specifically, we now note that strain QWL-01ᵀ is likely to function as a strictly anaerobic fermentative bacterium involved in the turnover of soluble organic matter in sludge, and that its genome suggests potential capacities related to oxygen tolerance, detoxification, and pollutant transformation. We also added a statement (Lines 472-480) that these features may make this strain a useful microbial resource for future studies on anaerobic sludge microbiology and potential biotechnological applications, while acknowledging that these functional inferences remain to be validated experimentally.]

Reviewer 4 Report

Comments and Suggestions for Authors

This article has scientific potential, and the taxonomy proposal appears credible; but, the present version requires revision. My primary concerns are to the clarity of taxonomic reasoning at the genus level, the accuracy of some methodological descriptions, and the need for meticulous language and editorial correction. These are revisable issues rather than fatal flaws. I therefore recommend revision before the manuscript can be considered for acceptance.

The following specific comments are provided below.

Line 4: “waste water” should be standardized to “wastewater”.
Line 38: The phrase "Analysis of the Biolog AN plate reveals" shoud be written in the past tense.
Line 53: The keywords are acceptable, but they could be improved by including terms more directly reflecting the taxonomic nature of the work, such as such as "Clostridiaceae," "polyphasic taxonomy," or "novel genus".
Lines 56–77: The introduction should be more focused by connecting the background more directly to the taxonomic isolation of novel anaerobes from engineered sludge environments, making it clear why culture-based isolation is still important even though metagenomic advances have been made, and briefly explaining why strain QWL-01T was chosen for further study.
Lines 82–83: NH4Cl appears to be listed twice in the composition.
Lines 97–106: Please explain if the first enrichment was done in duplicate, if serial dilution was done before each rolling-tube purification round, and how purity was checked after each round instead of just at the end.
Line 100: Please fix the sentence that states "prepared according to the instructions of the medium".
Lines 117–123: Please be more specific about how you describe these conditions and make sure that the language used throughout the text is uniform.
Lines 176–178: Please don't use website names as the only part of a sentence. It would read more scientifically to state the methods and then cite the corresponding tools and references more smoothly.
Line 185: "bioinformatics" rather than "bioinfomatics".
Line 191: There seems to be a mistake in "China Center of Industrial Culture Collection, Chain."
Line 208: “light and electronic microscopes” in the caption should be revised
Lines 218–224: The way oxygen-related physiology is presented in the paper has to be carefully considered. Growth below the oxic layer in thioglycollate medium indicates tolerance to low-oxygen exposure rather than aerobic growth. This distinction should be made explicit.
Lines 243–252: The authors should mention more explicitly that the values are consistent with genus-level separation rather than leaving the reader to infer this from the numbers alone.
Lines 263–273: This section might benefit from a more cohesive concluding statement showing why the combined use of ANI, dDDH, AAI, and phylogenomic findings support the proposition of a new genus rather than just a unique species.
Line 290: "the gram-positive structure" should be revised.
Lines 306–308: Please avoid overinterpreting the presence of oxidative stress genes as direct proof of physiological function without qualification.
Lines 309–312: The conclusion is very brief.
Lines 313–331: Please check punctuation, italicization, and typographic consistency carefully, as taxonomic descriptions require particularly high editorial precision.
The authors should meticulously verify the journal formatting, capitalization, and style consistency. A few entries exhibit style errors.
The order of figure appearance should be corrected. In the current version, Figure 2 appears before Figure 1. Please ensure that figures are cited and presented sequentially in the main text, in accordance with Microorganisms MDPI style requirements.
Table 1 includes six strains in total, whereas Table 2 includes only five, without explanation. In addition, the reference support for some previously described strains differs between the tables; for example, P. ruminis JCM 14817T is cited as [39] in Table 1 but as [39,46] in Table 2.

Comments on the Quality of English Language

The manuscript is generally comprehensible; however, the English language requires improvement in terms of clarity, grammar, and scientific style. There are several sentences that are awkwardly phrased, and certain sections could benefit from meticulous language refining to enhance precision and readability.

Author Response

Thank you for your detailed revisions. All revised parts have been highlighted in yellow for ease of reference.

Reviewer 4

The following specific comments are provided below.

Comments 4-1: [Line 4: “waste water” should be standardized to “wastewater”.]

Responses 4-1: [Thank you for your careful revision. In line 4, we replaced “waste water” with “wastewater.”.]

Comments 4-2: [Line 38: The phrase "Analysis of the Biolog AN plate reveals" should be written in the past tense.]

Responses 4-2: [Thank you for your careful revision. In line 39, we replaced “reveals” with “revealed”.]

Comments 4-3: [Line 53: The keywords are acceptable, but they could be improved by including terms more directly reflecting the taxonomic nature of the work, such as such as "Clostridiaceae," "polyphasic taxonomy," or "novel genus".]

Responses 4-3: [Thank you for your suggestions. In Lines 53–54, we replaced “Steel factory” with “Novel genus” and “Anaerobe” with “Clostridiaceae”.]

Comments 4-4: [Lines 56–77: The introduction should be more focused by connecting the background more directly to the taxonomic isolation of novel anaerobes from engineered sludge environments, making it clear why culture-based isolation is still important even though metagenomic advances have been made, and briefly explaining why strain QWL-01T was chosen for further study.]

Responses 4-4: [Thank you for this helpful suggestion. We agree that the Introduction should more clearly connect the general background to the isolation and taxonomic characterization of novel anaerobes from engineered sludge environments. In the revised manuscript, we added a paragraph to emphasize that cultivation-based isolation remains essential despite recent advances in metagenomics, and that small-scale culturomics provides an effective strategy for recovering novel anaerobic taxa from complex sludge systems (Lines 66-87). We also clarified why strain QWL-01ᵀ was selected for further study, namely because it was recovered as a distinct anaerobic isolate from sewage sludge and showed clear taxonomic novelty during the initial phylogenetic characterization (Lines 101-104). These revisions make the rationale of the study and the choice of strain QWL-01ᵀ more explicit.]

Comments 4-5: [Lines 82–83: NH4Cl appears to be listed twice in the composition.]

Responses 4-5: [Thank you for your detailed revision. In the original line 83: deleted “NH₄Cl, 1.0 g;”.]

Comments 4-6: [Lines 97–106: Please explain if the first enrichment was done in duplicate, if serial dilution was done before each rolling-tube purification round, and how purity was checked after each round instead of just at the end.]

Responses 4-6: [Thank you for this helpful comment. We have clarified the purification procedure in the revised manuscript. The initial enrichment was not performed in duplicate. However, serial dilution was carried out before each round of rolling-tube purification to facilitate the isolation of single colonies. After each purification round, the isolate was checked by 16S rRNA gene sequencing to confirm that the same bacterial strain had been recovered, and purity was also assessed at each round before proceeding to the next purification step. The relevant description has been revised in the Materials and Methods section to make these procedures clearer (Lines 131-139).]

Comments 4-7: [Line 100: Please fix the sentence that states "prepared according to the instructions of the medium".]

Responses 4-7: [Thank you for your comment. The original sentence has been revised as “Approximately 2 kg of sludge was transported to the laboratory in a plastic bag within 2 h of sampling, and about 3 mL of sludge, collected using a syringe, was inoculated into anaerobic modified DSM 120 medium and incubated at room temperature (~25 °C) for 2 weeks” (Lines 128-131) according to the reviewer’s comments.]

Comments 4-8: [Lines 117–123: Please be more specific about how you describe these conditions and make sure that the language used throughout the text is uniform.]

Responses 4-8: [Thank you for this helpful comment. We agree that the description of these growth conditions should be more specific and that the terminology should be used more consistently throughout the manuscript. In the revised manuscript, we clarified that aerobic growth on solid medium was assessed by incubation in ambient air, whereas growth under low-oxygen conditions was evaluated in thioglycollate medium under static incubation to establish an oxygen gradient. We also revised the wording in this section to ensure more uniform language throughout the text (Lines 152-161).]

Comments 4-9: [Lines 176–178: Please don't use website names as the only part of a sentence. It would read more scientifically to state the methods and then cite the corresponding tools and references more smoothly.

Responses 4-9: [Thank you for this helpful comment. In the revised manuscript, we rephrased the relevant sentences to describe the analytical methods first and then cited the corresponding tools, databases, and references in a smoother manner (Lines 207-212). These changes improve the clarity and scientific style of the Methods section.]

Comments 4-10: [Line 185: "bioinformatics" rather than "bioinfomatics".]

Responses 4-10: [Thank you for your careful revision. In the line 218, we replaced “bioinfomatics” with “bioinformatics”.]

Comments 4-11: [Line 191: There seems to be a mistake in "China Center of Industrial Culture Collection, Chain."]

Responses 4-11: [Thank you for your careful revision. In the line 224, we replaced “Chain” with “China”.]

Comments 4-12: [Line 208: “light and electronic microscopes” in the caption should be revised]

Responses 4-12: [Thank you for your comment. We have revised the original sentence as “Micrographs of strain QWL-01ᵀ obtained by light microscopy, transmission electron microscopy, and scanning electron microscopy.”]

Comments 4-13: [Lines 218–224: The way oxygen-related physiology is presented in the paper has to be carefully considered. Growth below the oxic layer in thioglycollate medium indicates tolerance to low-oxygen exposure rather than aerobic growth. This distinction should be made explicit.]

Responses 4-13: [Thank you for this helpful comment. In the revised manuscript, we have revised the relevant text to make this distinction explicit. Aerobic growth is now described only on the basis of incubation in ambient air on solid medium, whereas growth in thioglycollate medium is interpreted as evidence of tolerance to low-oxygen conditions.

In the new lines 251–256, we revised the original sentence as “Strain QWL-01ᵀ did not form colonies on DSM 120 agar plates under aerobic conditions. However, growth was observed beneath the red (oxic) layer in thioglycollate (TGC) medium, indicating tolerance to limited oxygen exposure or growth under low-oxygen conditions rather than true aerobic growth. This observation is consistent with the presence of oxygen tolerance-related genes identified in the genome of strain QWL-01ᵀ (see Section 4.3).”

We also revised the Discussion accordingly to avoid overinterpretation of the oxygen-related physiology of strain QWL-01ᵀ.

In the new lines 358–361, we revised the original sentence as “Although strain QWL-01ᵀ did not grow under aerobic conditions, growth was observed beneath the oxic layer in TGC medium, indicating tolerance to limited oxygen exposure or the ability to persist under low-oxygen conditions rather than true aerobic growth.”]

Comments 4-14: [Lines 243–252: The authors should mention more explicitly that the values are consistent with genus-level separation rather than leaving the reader to infer this from the numbers alone.]

Comments 4-15: [Lines 263–273: This section might benefit from a more cohesive concluding statement showing why the combined use of ANI, dDDH, AAI, and phylogenomic findings support the proposition of a new genus rather than just a unique species.]

Responses to comments of 4-14 and 4-15: [Thank you for this helpful comment. We agree that this section would benefit from a more cohesive concluding statement. In the revised manuscript, we added a concluding sentence to explicitly integrate the ANI, dDDH, AAI, and phylogenomic results, and to clarify that these combined genomic data support the proposal of strain QWL-01ᵀ as representing a novel genus rather than a unique species within an existing genus.

In the new lines 309-328, we have revised the original sentences as “To further clarify the taxonomic position……Since both the 16S rRNA gene sequence similarities and the AAI values are below the commonly accepted genus-level reference ranges, strain QWL-01ᵀ is more appropriately classified as representing a novel genus rather than a novel species within either Youngiibacter or Proteiniclasticum.”]

Comments 4-16: [Line 290: "the gram-positive structure" should be revised.]

Responses 4-16: [Thank you for this comment. We revised “the gram-positive structure” as “a Gram-positive-type cell envelope structure” in the revised manuscript. And whole paragraph of section 4.1 has been revised as “Strain QWL-01ᵀ showed a Gram-stain-negative reaction under the conditions tested……Taken together, these results indicate that the Gram-stain-negative reaction of strain QWL-01ᵀ should be interpreted cautiously as a staining phenotype and does not contradict its genome-based taxonomic placement or its inferred monoderm cell envelope structure.” (Lines 331-349)]

Comments 4-17: [Lines 306–308: Please avoid overinterpreting the presence of oxidative stress genes as direct proof of physiological function without qualification.]

Responses 4-17: [Thank you for this helpful comment. Some revisions were made as follows.

In the new lines 364-368, we revised the original sentence “These enzymes are known to detoxify reactive oxygen species and maintain intracellular redox balance……. lacks the ability to grow under fully aerobic conditions.” as “These enzymes are generally associated with detoxification of reactive oxygen species and maintenance of intracellular redox balance. Therefore, the presence of the corresponding genes in the genome is consistent with a potential capacity of strain QWL-01ᵀ to tolerate transient or low levels of oxygen, although this function was not directly demonstrated experimentally in the present study.”]

Comments 4-18: [Lines 309–312: The conclusion is very brief.]

Responses 4-18: [Thank you for this helpful comment. In the revised manuscript, we expanded the Conclusion to better summarize the main taxonomic findings as well as the possible ecological and functional relevance of strain QWL-01ᵀ.

In the new lines 472-480, we added a paragraph of “Beyond its taxonomic novelty, phenotypic and genome-based evidence suggests that this strain is a strictly anaerobic fermentative bacterium……However, these proposed functional roles remain to be validated experimentally” to expand the conclusion.]

Comments 4-19: [Lines 313–331: Please check punctuation, italicization, and typographic consistency carefully, as taxonomic descriptions require particularly high editorial precision.]

Responses 4-19: [Thank you for this helpful comment. We agree that taxonomic descriptions require a high level of editorial precision. In the revised manuscript, we consulted an ICSP member and carefully reviewed the entire text for punctuation, italicization, and typographic consistency, with particular attention to the taxonomic descriptions and the formatting of scientific names. We corrected the gender of the genus name Anaerococcoides to neuter and accordingly revised the species epithet from asporogenes to asporogena, in compliance with the current nomenclatural rules for generic names ending in -oides. These changes were applied consistently throughout the manuscript, including the title, abstract, taxonomic descriptions, and all other occurrences of the species name.]

Comments 4-20: [The authors should meticulously verify the journal formatting, capitalization, and style consistency. A few entries exhibit style errors.]

Responses 4-20: [Thank you for this helpful comment. In the revised manuscript, we thoroughly reviewed the entire text and corrected the identified style inconsistencies, including capitalization, italicization, punctuation, formatting of scientific names, section headings, references to figures and tables, and the presentation of type strain designations. These changes were applied throughout the manuscript to improve consistency with the journal’s formatting and style requirements.]

Comments 4-21: [The order of figure appearance should be corrected. In the current version, Figure 2 appears before Figure 1. Please ensure that figures are cited and presented sequentially in the main text, in accordance with Microorganisms MDPI style requirements.]

Responses 4-21: [Thank you for this helpful comment. We agree that the figures should be cited and presented in sequential order. In the revised manuscript, we carefully checked the order of figure citation in the main text and corrected it so that Figure 1 now appears before Figure 2, in accordance with the Microorganisms formatting requirements. We also reviewed the remaining figure citations to ensure consistency throughout the manuscript.]

Comments 4-22: [Table 1 includes six strains in total, whereas Table 2 includes only five, without explanation. In addition, the reference support for some previously described strains differs between the tables; for example, P. ruminis JCM 14817T is cited as [39] in Table 1 but as [39,46] in Table 2.]

Responses 4-22: [Thank you for this helpful comment. We agree that both the discrepancy in the number of strains compared in Tables 1 and 2 and the inconsistency in reference citation required clarification. In the revised manuscript, we have added an explanation (Lines 277-279) that Table 2 includes only five strains because detailed fatty acid composition data for Youngiibacter multivorans DSM 6139ᵀ were not available for direct comparison. We also reviewed and standardized the reference citations throughout both tables, including those for Proteiniclasticum ruminis JCM 14817ᵀ. These corrections improve the transparency and internal consistency of the manuscript.]

Round 2

Reviewer 2 Report

Comments and Suggestions for Authors

The revised manuscript effectively addresses all previous concerns, and the updates have improved clarity.
The manuscript is suitable for publication in its current form, and I recommend its acceptance.

Reviewer 3 Report

Comments and Suggestions for Authors

To my opinion, the authors have significantly revised the manuscript and taken into account all my comments. I am ready to recommend the manuscript for publication in this form.