Bioleaching Process of Sewage Sludge and Anaerobically Digested Sludge via Indigenous Sulfur-Oxidizing Bacteria to Improve Dewaterability and Reduce Heavy Metal Content

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

This manuscript has good novelty and relatively rich experimental results. However, the manuscript should not be published in its current form.

Specific comments:

1. Title, “land application” is too broad. It is suggested to adopt more specific words (such as dewaterability,heavy metals).
2. Please verify the author's information.
3. It is suggested that the repetition rate of the thesis be controlled below 20%.
4. Line 58-61, this part should be elaborated in more detail.
5. Line 71-83, It is suggested to split it into two sections (Line 71-77 and 77-83)
6. Table 1. standard deviation should be provided.
7. Figure 2, error bar should be clearer (Part of it has been covered).
8. Line 193, Please check the content carefully.
9. The form of the figure should be uniform (Such as Figure 2, Figure 3, Figure 4).
10. Figure 4, It is suggested to merge Figure a and Figure b.
11. Figure 5, Because the solid content of S and ADS is different. It is suggested that the CST value be normalized according to the sludge concentration.
12. Figure 6, It is suggested to be expressed based on volatile solids.
13. Table 2, It is suggested to present it in the form of a figure.
14. Anaerobic digested sludge has a relatively high alkalinity, it may affect the change of pH. If possible, please provide the alkalinity information of the sample.
15. Line 303-304， “dewaterabilty” is better than“dehydration performance”.
16. Conclusions, conclusions should be concise and powerful.

Author Response

1. Title, “land application” is too broad. It is suggested to adopt more specific words (such as dewaterability, heavy metals).

Response: Thanks for the suggestion. It intends emphasize the improvement of dewatering performance of sludge and the reduction of the risk of heavy metal pollution after biological treatment. Thus, the title is changed to “Bioleaching process of sewage sludge and anaerobically digested sludge via indigenous sulfur-oxidizing bacteria to improve dewaterability and reduce heavy metal content”.

 

1. Please verify the author's information.

Response: Thanks for the suggestion and we've verified the author's information.

 

1. It is suggested that the repetition rate of the thesis be controlled below 20%.

Response: Thanks for the suggestion and we've reduced the duplication rate, which is as follows:

Line 25-27: “While the S/ADS+B configuration exhibited marginally reduced Cu, Ni, and Pb removal efficiencies relative to S/ADS+E, it demonstrated superior dewaterability characteristics under equivalent reaction durations.”

Line 34-36: “The management and final disposition of sewage sludge (SS) constitutes the most technologically challenging and cost-intensive component within wastewater treatment infrastructure.”

Line 36-37: “Sludge stabilization through anaerobic digestion represents the predominant approach for processing organic-rich sludge materials.”

Line 45-47: “The anaerobic digestion process induces degradation of dissolved extracellular polymeric substances (EPS), comprising proteins and polysaccharides, thereby compromising the dewatering characteristics of ADS.”

Line 53-54: “Contemporary research has identified bioleaching as an economically viable dual-purpose technology for simultaneous sludge dewaterability enhancement and heavy metal extraction.”

Line 79-80: “This metabolic complementarity explains the documented superiority of co-cultured heterotroph-autotroph systems over axenic cultures in mineral oxidation applications.”

Line 103-104: “Microbial cultivation occurred under controlled conditions (28°C thermostatic water bath, 180 rpm orbital agitation).”

Line 119-120: “Total suspended solids, volatile suspended solids, and DOC quantified following established analytical protocols.”

Line 171-172: “High-throughput sequencing elucidated structural modifications in microbial consortia during acclimatization culturing”

Line 179-180: “Acid-tolerant heterotrophs dominated the enriched consortium, comprising 52.7% of operational taxonomic units (OTUs).”

 

1. Line 58-61, this part should be elaborated in more detail.

Response: Thank you for your comment and we've added more detail, which is as follows:

Line 62-72: The precise biochemical pathways underpinning this synergistic enhancement of both sludge dewaterability and heavy‐metal removal in ADS are not yet fully elucidated. In particular, most acidophilic sulfur‐oxidizing bacteria operate strictly as autotrophs, deriving energy solely from inorganic substrates; any influx of organic carbon, such as low‐molecular‐weight peptides, amino acids, or polysaccharides released during cell lysis-increases the dissolved organic carbon (DOC) fraction in solution. Elevated DOC levels can complex with protonated metal ions, altering their speciation and redox potential, and may also form a protective colloidal layer on sludge particles, thereby inhibiting the attachment, biofilm formation, and sulfidogenesis activity of the acidophilic consortia. Together, these effects can diminish proton‐driven metal solubilization rates and reduce extracellular polymeric substance (EPS) degradation, ultimately dampening both dewatering performance and metal leaching efficiency.

 

1. Line 71-83, It is suggested to split it into two sections (Line 71-77 and 77-83)

Response: Thank you for your comment and we've split it into two sections.

 

1. Table 1. standard deviation should be provided.

Response: Thank you for your advice and we've provided standard deviation.

Table 1 Characteristics of fresh sewage sludge and anaerobically digested sludge.

Parameters	SS	ADS
pH	7.24±0.55	8.08±0.36
TS (%)	2.31±0.20	4.20±0.14
VS (%)	1.33±0.08	2.10±0.10
SCOD (mg/L)	110.0±4.67	1656.7±22.11
Ni (mg/kg TS)	147.8±3.26	50.5±1.94
Pb (mg/kg TS)	76.4±2.11	52.5±2.34
Cu (mg/kg TS)	431.9±14.84	786.3±24.13
Cr (mg/kg TS)	363.1±19.51	196.8±9.41

 

1. Figure 2, error bar should be clearer (Part of it has been covered).

Response: Thank you for your advice, this figure has be adjusted.

 

1. Line 193, Please check the content carefully.

Response: Thank you for your advice and we've revised this sentence.

 

1. The form of the figure should be uniform (Such as Figure 2, Figure 3, Figure 4).

Response: Thank you for your advice and we've revised these figures.

 

1. Figure 4, It is suggested to merge Figure a and Figure b.

Response: Thanks for the suggestion. Figure 4 has been revised.

 

1. Figure 5, Because the solid content of S and ADS is different. It is suggested that the CST value be normalized according to the sludge concentration.

Response: Thanks for the suggestion. The CST values are normalized by dividing them by the initial TS concentration and then expressed in units of s•L/gTS.

 

1. Figure 6, It is suggested to be expressed based on volatile solids.

Response: Thanks for the suggestion. The values of Fig. 6 are presented based on the volatile solids.

 

1. Table 2, It is suggested to present it in the form of a figure.

Response: Thanks for the suggestion. As limited data are utilized, thus this data is presented in Table.

 

1. Anaerobic digested sludge has a relatively high alkalinity, it may affect the change of pH. If possible, please provide the alkalinity information of the sample.

Response: Thank you for this valuable suggestion. Unfortunately, we did not measure alkalinity in our current set of experiments. We recognize that alkalinity can significantly influence pH dynamics, and we will include detailed alkalinity determinations (e.g., as mg CaCO₃/L) in our follow-up studies to clarify its role in pH changes.

 

1. Line 303-304， “dewaterabilty” is better than“dehydration performance”.

Response: Thanks for the suggestion and we've changed the “dehydration performance” to “dewaterabilty”.

 

1. Conclusions, conclusions should be concise and powerful.

Response: Thanks for the comment and we've changed the conclusions, which is as follows:

The bioleaching of SS and ADS by indigenous sulfur‐oxidizing consortia proved both highly effective and economical: under optimized conditions (10 % inoculum, limited S⁰), pH was stably maintained between 2.0–3.0, enabling >90 % removal of Ni and Pb and 30–70 % removal of Cu and Cr. Simultaneously, sludge dewaterability improved by up to 30 % (CST reduction) without chemical additives. Direct and indirect leaching pathways worked in concert to solubilize metals while moderate acidification prevented excessive EPS release and CST rebound. This one‐step, low‐cost approach holds strong promise for safe, resource‐efficient conditioning of both fresh and digested sludges.

 

Reviewer 2 Report

Comments and Suggestions for Authors

The work fermentation-3646280 is devoted to the study of the Bioleaching process of primary and secondary sewage sludge using a consortium of sulfur-oxidizing bacteria. The work is relevant, novel and has practical significance.

The merit of the work is the elucidation of the mechanism of regulation of the Bioleaching process by monitoring the concentration of sulfur in the environment. The work is performed at a good methodological level.

 

The elimination of the following comments will help improve the perception of the work:

1) Too many abbreviations. This is very distracting and interferes with perception.

2) The abstract is written incomprehensibly. It is impossible to understand from the abstract what E is and what B is.

3) In the introduction, the authors refer to their own works to justify the relevance of the task. This is not scientifically substantiated, because perhaps the authors convey only their opinion, and not the opinion of the world community on the issue of relevance. In the formulation of the problem, other people's works should be cited.

4) The review of achievements includes mainly old works. There are only 3 works from the last 5 years out of 18 references given in the introduction. Perhaps the task has already been solved by colleagues or competitors?

5) It should be emphasized that the local consortium of sulfur-oxidizing bacteria outperforms the work of individual strains.

Author Response

The merit of the work is the elucidation of the mechanism of regulation of the Bioleaching process by monitoring the concentration of sulfur in the environment. The work is performed at a good methodological level.

First and foremost, we sincerely thank you for your time and constructive feedback on our manuscript. We greatly appreciate the opportunity to revise our work and address the insightful comments provided. Below, we have carefully responded to each point raised, incorporating revisions to improve clarity, depth, and scientific rigor.

The elimination of the following comments will help improve the perception of the work:

1) Too many abbreviations. This is very distracting and interferes with perception.

Response: Thanks for pointing out this question, In the revised manuscript, we have removed or expanded all non-essential abbreviations to improve readability. Only the most common terms remain abbreviated (e.g. sewage sludge (SS), anaerobically digested sludge (ADS), extracellular polymeric substances (EPS), dissolved organic carbon (DOC), capillary suction time (CST), etc.) and each is defined when first introduced.

All other previously used shorthand (such as single-letter codes or unused acronyms) has been replaced by descriptive terms. For example, throughout the text we now consistently write “inoculum” or “energy source (elemental sulfur)” instead of “inoculum” (+B) or “S⁰” alone, except where the abbreviation (e.g. S⁰) is immediately defined. This substantially reduces confusion. No change in meaning has occurred; we have simply spelled out terms to make the text self-contained.

 

2) The abstract is written incomprehensibly. It is impossible to understand from the abstract what E is and what B is.

Response: We thank the reviewer for pointing out the ambiguity in the abstract. In the revised abstract we explicitly define these treatment codes. For example, we changed the description “ Four treatments were evaluated: inoculum + elemental sulfur (S/ADS+E), inoculum alone (S/ADS+B), elemental sulfur alone (S/ADS+S), and a control with no additives.” This revision makes clear that E refers to the treatment with elemental sulfur (energy source) plus inoculum, and B refers to the inoculum (bacteria) alone. All abbreviations are now defined on first use. For instance, we wrote “elemental sulfur (S⁰)” when first mentioning sulfur. This change resolves the confusion about “E” and “B” in the abstract.

3) In the introduction, the authors refer to their own works to justify the relevance of the task. This is not scientifically substantiated, because perhaps the authors convey only their opinion, and not the opinion of the world community on the issue of relevance. In the formulation of the problem, other people's works should be cited.

Response: Thanks for the comment and we have included recent investigations of microbial consortia in heavy-metal remediation. For example, we cite Khidr et al. (2025, Environ. Geochem. Health) who demonstrated that co-cultures of indigenous bacteria achieved significantly higher metal reduction (≈47–62% removal of Cu, Ni, Zn) than any single strain. We also mention other contemporary works (e.g. Sustainable heavy metal removal from sludge , Molaey et al. 2024) that discuss state-of-the-art bioleaching strategies and de-metallization techniques. These additions ensure that at least half of the introduction’s key references are from the last five years, providing up-to-date context. For brevity, only a sample of the revised text is shown here; the revised Introduction now reads:

Line 57-62: “Bioleaching of sewage sludge has been shown to efficiently remove heavy metals under mild conditions [10]. Concurrently, bioleaching often enhances sludge dewaterability (e.g. Shokoohi et al. 2025 report a 66.9% decrease in sludge filtration resistance after bioleaching [11]). Recent work also highlights the role of microbial consortia: Khidr et al. (2025) found that mixed indigenous cultures outperformed monocultures, achieving metal removal efficiencies of up to ~62% [12].”

4) The review of achievements includes mainly old works. There are only 3 works from the last 5 years out of 18 references given in the introduction. Perhaps the task has already been solved by colleagues or competitors?

Response: Thanks for the comment and we have addressed this by updating and enriching the literature review with recent references from 2020–2025. Which is as follows:

10. Li, T., Yang, J., Zhou, Y., Luo, Y., Zhou, B., Fang, D., Li, J., Zhou, L. Enhancing sludge dewatering efficiency through bioleaching facilitated by increasing reactive oxygen species. Water research, 231, 119622, 2023.
11. Shokoohi, R., Najafi-Vosough, R., Shabanloo. Removing heavy metals and improving the dewaterability of sewage sludge with the bioleaching process by Thiobacillus Ferrooxidans bacteria. Applied Water Science, 15, 34, 2025.
12. Khidr, R., Qurbani, K., Muhammed, V., Salim, S., Abdulla, S., & Wsw, H. . Synergistic effects of indigenous bacterial consortia on heavy metal tolerance and reduction. Environmental Geochemistry and Health, 47(3), 79, 2025.
13. Wang, Z., Feng, W., Tang, S., Zhao, J., Zheng, G., Zhou, L, Enhancing sludge dewaterability in sequential bioleaching: Degradation of dissolved organic matter (DOM) by filamentous fungus Mucor sp. ZG-3 and the influence of energy source. PloS one, 19(5), e0302311, 2024.

These studies, along with others from Water Research and Environmental Sci. & Tech., are now incorporated to substantiate the relevance of using indigenous sulfur-oxidizers and to situate our research within the current state of the field.”

 

5) It should be emphasized that the local consortium of sulfur-oxidizing bacteria outperforms the work of individual strains.

Response: Thanks for the comment. We reference the sequential bioleaching study by Wang et al. (2024), where adding a fungal pretreatment to A. ferrooxidans alone reduced sludge filtration resistance by 25.9% (relative improvement), illustrating the advantage of a mixed process over a single-strain approach.

Line 345-350: The added text now reads: “Recent studies demonstrate the superiority of indigenous consortia: for instance, Khidr et al. found that co-cultures of native strains greatly exceeded individual strains in heavy-metal reduction [12, 44]. Similarly, our work leverages the natural sulfur-oxidizing community rather than a single purified strain, which is expected to enhance robustness and metal solubilization (consistent with the improved outcomes seen in consortium-based bioleaching)”.

Round 2

Reviewer 1 Report

Comments and Suggestions for Authors

After revisions, I give the green light to this manuscript.