Coupling Granular Activated Carbon with Waste Iron Scraps Enhances Anaerobic Digestion of PBAT Wastewater: Performance Improvement and Mechanistic Insights

Response to Reviewer X Comments

1. Summary

We have carefully revised the manuscript according to all suggestions made by the editor and the reviewers. A detailed description of the revision and our replies to the reviewers’ comments on a point-by-point base can be found in the following “Response to editor and reviewers”. We have carefully checked all author names and corrected exactly the style of the references and formatting according to the journal instructions.

We have used professional language editing options to check our manuscript for grammar, style, and syntax. The revised manuscript looks much better than the original one, and we would like to take this opportunity to thank the editors for their valuable comments.

Thank the reviewers for their valuable comments and we have modified and replied point by point.

Thank you for your time and consideration.

2. Questions for General Evaluation

Reviewer’s Evaluation

Response and Revisions

Does the introduction provide sufficient background and include all relevant references?

Yes/Can be improved/Must be improved/Not applicable

I will give corresponding responses in each reply letter.

Are all the cited references relevant to the research?

Yes/Can be improved/Must be improved/Not applicable

Is the research design appropriate?

Yes/Can be improved/Must be improved/Not applicable

Are the methods adequately described?

Yes/Can be improved/Must be improved/Not applicable

Are the results clearly presented?

Yes/Can be improved/Must be improved/Not applicable

Are the conclusions supported by the results?

Yes/Can be improved/Must be improved/Not applicable

3. Point-by-point response to Comments and Suggestions for Authors

Comments 1: Please clarify; in the paragraph there is a reference to waste and in the next sentence to effluent.Line 35-39 „The process generates highly concentrated and acidic organic waste streams, characterized by a chemical oxygen demand (COD) of 10–20 g/L and 36 a pH ranging from 3.5 to 5.5 [3,4]. This effluent contains high concentrations of unreacted 37 monomers, oligomers, and other biotoxic substances, which presents a substantial chal-38 lenge to conventional biological treatment systems [5]”.

Response 1: Thank you for the valuable comments. In lines 35-39 of the original text, both "waste streams" and "effluent" refer to the organic wastewater generated during the production process of PBAT, and their core meanings are consistent. We have replaced "waste streams" in the text with "wastewater" to ensure the smooth continuation of "effluent" in the following text and guarantee the unity and clarity of the concepts throughout the text.

Comments 2: Since PBAT is the basic substrate for research, despite the literature source provided, please specify the preparation of this wastewater for research. Line 93, 93 „The PBAT  wastewater was prepared artificially, with its composition and water quality parameters 93 determined according to previous studies [21]”.

Response 2: Thank you for the valuable comments. Line 93 of the original text only mentions "prepared based on previous research [21]", which is rather brief. Now, in combination with the actual operation of this study and the core parameters of previous research [21], the detailed components and water quality parameters of artificial water preparation can be found in Table 1 and Table 2.”Table 1. Water quality parameters of PBAT simulated wastewater. Table 2. Main components of artificial water distribution.”

Comments 3: The conclusions are too general; please relate them to the specific research objectives. We have revised

Response 3: Thank you for the valuable comments. In line with the research objectives, we have revised the conclusion. The modified content is as follows: ”This study verified the synergistic effects of GAC and WIS in enhancing the anaerobic digestion of PBAT wastewater. Compared to individual additions, the coupling of GAC and WIS significantly improved methane production and COD removal efficiency. The underlying mechanisms were that GAC effectively reduced the inhibitory toxicity of refractory organics through adsorption and microbial enrichment, while WIS buffered the system pH by the release of Fe²⁺ and H₂. Coupling GAC and WIS effectively mitigated the accumulation of VFAs and promoted the transformation of complex organic matter. Furthermore, the favorable microenvironment synergistically enhanced the activity of both hydrogenotrophic and acetoclastic methanogenic pathways and significantly improved the DIET efficiency among microbial communities. Finally, analysis of metagenomic sequencing indicated that GAC enriched electro-active fermentative bacteria and methanogens, whereas WIS stimulated the growth of hydrogenotrophic methanogens; coupling GAC and WIS synergistically drove the functional succession of the microbial community and the upregulation of key metabolic genes. This study provided a theoretical foundation and engineering strategy for the treatment of PBAT wastewater.”

4. Response to Comments on the Quality of English Language

Point 1: The English is fine and does not require any improvement.

Response 1: Thank you for your affirmation of me.

5. Additional clarifications

No.

Response to Reviewer X Comments

1. Summary

We have carefully revised the manuscript according to all suggestions made by the editor and the reviewers. A detailed description of the revision and our replies to the reviewers' comments on a point-by-point base can be found in the following "Response to editor and reviewers". We have carefully checked all author names and corrected exactly the style of the references and formatting according to the journal instructions.

We have used professional language editing options to check our manuscript for grammar, style, and syntax. The revised manuscript looks much better than the original one, and we would like to take this opportunity to thank the editors for their valuable comments.

Thank the reviewers for their valuable comments and we have modified and replied point by point.

Thank you for your time and consideration.

2. Questions for General Evaluation

Reviewer's Evaluation

Response and Revisions

Does the introduction provide sufficient background and include all relevant references?

Yes/Can be improved/Must be improved/Not applicable

I will give corresponding responses in each reply letter.

Are all the cited references relevant to the research?

Yes/Can be improved/Must be improved/Not applicable

Is the research design appropriate?

Yes/Can be improved/Must be improved/Not applicable

Are the methods adequately described?

Yes/Can be improved/Must be improved/Not applicable

Are the results clearly presented?

Yes/Can be improved/Must be improved/Not applicable

Are the conclusions supported by the results?

Yes/Can be improved/Must be improved/Not applicable

3. Point-by-point response to Comments and Suggestions for Authors

Comments 1: Abstract:

1) "Batch experiments demonstrated that the GAC/WIS increased COD removal by 5.48-fold and methane production by 12.14-fold compared to the control, while reducing VFAs accumulation by 98.48%." The increase in multiples is amazing, but it should be marked to indicate how much the data has increased, and it is meaningful to match the revelation of the data.

2) "This study provided a cost-effective, sustainable strategy for PBAT wastewater treatment." What were the meanings of cost-effective and sustainable strategy?

Response 1: Thank you for the valuable comments. According to the comments, we have revised the abstract. The modified content is as follows: 1)"Poly(butylene adipate-co-terephthalate) (PBAT) wastewater, characterized by high chemical oxygen demand (COD) and acidity, poses significant challenges to anaerobic digestion (AD) due to toxicity and volatile fatty acids (VFAs) accumulation. This study coupled granular activated carbon (GAC) and waste iron scraps (WIS) to synergistically enhance AD performance. Batch experiments demonstrated that, compared with the control, the GAC/WIS group achieved a COD removal efficiency of 53.18% and a methane production of 207.53 ± 5.80 mL/g COD, which were 5.48- and 12.14-fold increases, respectively, while reducing the accumulation of total VFAs by 98.48% (to 15.09 mg/L). Mechanistic analysis revealed that GAC adsorbed inhibitors and enriched methanogens, while WIS buffered pH and promoted direct interspecies electron transfer (DIET) by hydrogenotrophic methanogenesis. Metagenomic sequencing showed shifts in microbial communities, with enrichment of syntrophic bacteria (Syntrophobacter) and functional genes (pta, bcd, pccA), indicating metabolic reprogramming. This study provided a theoretical foundation and engineering strategy for the treatment of PBAT wastewater."

Comments 2: Introduction:

Line 81-83: "This study provides novel insights and a theoretical foundation for the efficient and sustainable treatment of PBAT wastewater." This statement was not appropriate in the introduction.

Response 2: Thank you for the valuable comments. According to the comments, we have adjusted the introduction. The modified content is as follows:" GAC offers advantages in adsorption, bio-enrichment, and promoting DIET. WIS provides complementary roles in pH buffering, metabolic stimulation, enhancing specific methanogenic pathways, and mediating DIET. Therefore, it is hypothesized that coupling GAC and WIS could generate synergistic effects, which would address the dual challenges of toxicity and acidity inhibition in PBAT wastewater treatment more comprehensively and efficiently. Therefore, this study aimed to systematically evaluate the effects of GAC and WIS on the anaerobic digestion of PBAT wastewater. The specific objectives were: (1) To check the synergistic effects of GAC and WIS on enhancing methane production and COD removal efficiency. (2) To investigate the potential effects GAC and WIS on mitigating the accumulation of VFAs and promoting the degradation of recalcitrant organic matters. (3) To investigate the effects of GAC and WIS on the methanogenic activity and electron transfer activity. (4) To analyze the shifts in microbial community structure and functional gene expression that underpin the enhanced performance of the AD process. ”

Comments 3: Was there any research on the impact of WIS on pH improvement?

Response 3: Thank you for the valuable comments. References [20] and [21] investigated the effect of WIS on pH improvement. In . In reference [20], in 2.1.3 pH adjustment, it is mentioned that adding ZVI to anaerobic granular sludge can increase the pH value and maintain it at the initial value of 6.0-6.5. In reference [21], the results of 3.1 indicated that WIS significantly enhanced alkalinity and alleviated the inhibitory effect of low pH values on microorganisms.

Comments 4: Materials and Methods:

· What were the acetoclastic methanogenic activity and the hydrogenotrophic methanogenic activity? Were they obtained from sludge?

· The composition and water quality parameters of PBAT wastewater were very important. They should be revealed. It was not appropriate to self-cite previous works.

Response 4: Thank you for the valuable comments. Acetoclastic methanogenic activity refers to the methane production by acetoclastic methanogens in an anaerobic environment, with acetate as the substrate. Hydrogenotrophic methanogenic activity refers to the methane production by hydrogenotrophic methanogens in an anaerobic environment, with hydrogen (H₂) and carbon dioxide (CO₂) as the sole substrates. After four cycles of the batch experiments, 50 mL of anaerobic sludge was withdrawn from each bottle. The sludge in the serum bottles of each group was divided into two parts, one part for the determination of acetoclastic methanogenic activity and the other part for the determination of hydrogenotrophic methanogenic activity. Sodium acetate was used as the sole carbon source in the determination of acetoclastic methanogenic activity, while H₂/CO₂ (80%/20%) was used as the sole carbon source in the determination of hydrotropic methanogenic activity. The composition and water quality parameters of PBAT wastewater are shown in Tables 1 and 2.

Comments 5: The results of this study from the figures to demonstrate that coupling GAC and WIS synergistically enhanced the anaerobic digestion of PBAT wastewater, achieving significant improvements in COD removal and methane production. However, it is a pity that the detailed purpose and content of the experimental design cannot be known from the manuscript, and the logic and correlation between each experimental design cannot be known.

Response 5: Thank you for the valuable comments. In order to verify the synergistic effect of GAC and WIS on the anaerobic digestion of PBAT wastewater, a batch experiment was designed in Table 3. According to Table3, there were four groups,which were Control ,GAC, WIS and GAC/WIS group, respectively. The control group was the baseline group, which aimed to reflect the natural anaerobic digestion performance of PBAT wastewater without any additions and provided the reference for evaluating the enhanced effects of GAC, WIS and GAC/WIS. GAC group,which was added 20 g/L GAC, was aimed to verify the independent effect of GAC; WIS group,which was added 20 g/L WIS, was aimed to verify the independent effect of WIS. GAC/WIS group, which was added 10 g/L GAC + 10 g/L WIS, was aimed to verify the coupling effect of GAC and WIS in anaerobic digestion. The total mass of functional materials was consistent with that in the single-factor group (20 g/L), eliminating the influence of "difference in substance dosage" on the results. The aim was to test whether the combination of GAC (adsorption + microbial enrichment) and WIS (pH buffering + hydrogen-promoting trophication) can produce a "1+1>2" synergistic effect. In addition, to verify the long-term stability of the treatment effect, the experiment was conducted for four consecutive cycles (each cycle lasting 14 days). This is because industrial wastewater treatment requires long-term stable operation, and a single cycle may not reflect the persistence of the material's effect (such as whether GAC will reach adsorption saturation or whether WIS will be passivated). The four-cycle design ensured that the proposed strategy has practical application potential in industrial scenarios.

4. Response to Comments on the Quality of English Language

Point 1: The English is fine and does not require any improvement.

Response 1: Thank you for your affirmation of me.

5. Additional clarifications

No.

Response to Reviewer X Comments

1. Summary

We have carefully revised the manuscript according to all suggestions made by the editor and the reviewers. A detailed description of the revision and our replies to the reviewers' comments on a point-by-point base can be found in the following "Response to editor and reviewers". We have carefully checked all author names and corrected exactly the style of the references and formatting according to the journal instructions.

We have used professional language editing options to check our manuscript for grammar, style, and syntax. The revised manuscript looks much better than the original one, and we would like to take this opportunity to thank the editors for their valuable comments.

Thank the reviewers for their valuable comments and we have modified and replied point by point.

Thank you for your time and consideration.

2. Questions for General Evaluation

Reviewer's Evaluation

Response and Revisions

Does the introduction provide sufficient background and include all relevant references?

Yes/Can be improved/Must be improved/Not applicable

I will give corresponding responses in each reply letter.

Are all the cited references relevant to the research?

Yes/Can be improved/Must be improved/Not applicable

Is the research design appropriate?

Yes/Can be improved/Must be improved/Not applicable

Are the methods adequately described?

Yes/Can be improved/Must be improved/Not applicable

Are the results clearly presented?

Yes/Can be improved/Must be improved/Not applicable

Are the conclusions supported by the results?

Yes/Can be improved/Must be improved/Not applicable

3. Point-by-point response to Comments and Suggestions for Authors

Comments 1: Where were the waste iron scraps obtained from and what particle sizes were used?

Response 1: Thank you for the valuable comments. The waste iron scraps are from Longxin Water Purification Materials in Gongyi City, with a diameter of 0.3 to 0.7 cm. The modified content is as follows:" GAC (0.5-1.0 mm) and WIS (0.3-0.7 cm) were used, which were purchased from Sinopharm Chemical Reagent Co., Ltd and Gongyi Longxin Water Purification Materials Co., Ltd, respectively. "

Comments 2: For the granular activated carbon what were the particle sizes used? 

Response 2: Thank you for the valuable comments. The scrap iron filings are from Sinopharm Group, with a diameter of 0.5 to 1.0 mm. The modified content is as follows:" GAC (0.5-1.0 mm) and WIS (0.3-0.7 cm) were used, which were purchased from Sinopharm Chemical Reagent Co., Ltd and Gongyi Longxin Water Purification Materials Co., Ltd, respectively. ”

Comments 3: In Methods, Why dark conditions for the AD?

Response 3: Thank you for your valuable comments. Dark conditions were adopted for the AD process to avoid the inhibitory effects of light on methanogens. Methanogens (such as Methanothrix and Methanobacterium) are strictly anaerobic microorganisms, and key enzymes within their cells (such as methyl-coenzyme M reductase, encoded by mcrA) are sensitive to light exposure. Maintaining dark conditions eliminates light interference with these functional microorganisms, thereby ensuring the stable and efficient progression of methane production.

Comments 4: Was the volume of the biogas measures?

Response 4: Thank you for the valuable comments. According to the comments, According to the comments, we have added a method for measuring methane volume in the manuscript."Biogas production was measured volumetrically using calibrated gas-tight glass syringes (100 mL, Returnable, Shanghai, China). The composition of biogas, primarily methane (CH₄) and carbon dioxide (CO₂), was analyzed using a gas chromatograph (GC, SP-6890, Shandong Ruihong, China). The calculation formula for the final methane yield (mL/g COD) is: Methane yield = Cumulative volume of biogas volume fraction of methane / total COD consumed.

Comments 5: For the removal of COD and VAFs: it seems WIS and GAC acted as an adsorber?

Response 5: Thank you for the valuable comments. GAC reduced wastewater toxicity through the physical adsorption of inhibitory substances. Due to the conductive properties, GAC also facilitated direct interspecies electron transfer (DIET), thereby accelerating the conversion of VFAs into methane. In fact, after four cycles of operation, the adsorption of GAC has reached saturation. The primary function of GAC was to serve as a microbial attachment site, promoting biofilm formation and enriching specialized bacterial consortia capable of degrading recalcitrant organic compounds, which in turn enhanced COD removal. In parallel, WIS alleviated the inhibitory effect of low pH on hydrolytic bacteria, promoting COD degradation in PBAT wastewater and reducing VFAs accumulation. The coupling of GAC and WIS acted synergistically to improve the overall removal efficiency of COD and VFAs. The modified content is as follows: "The superior performance of the GAC/WIS combination, evidenced by a 5.48-fold increase in COD removal and a 12.14-fold increase in methane production compared to the Control group, highlighted a clear synergistic effect that addressed multiple limiting factors in PBAT wastewater anaerobic digestion. Interestingly, the GAC group showed the highest COD removal (79.0%) at the beginning (end of stage 1), but this was followed by a significant decline, likely due to the gradual saturation of the adsorption sites after initial efficient uptake. Despite the initial limitation of GAC when used alone, the enhanced and sustained performance of the combined system originated from the strategic coupling of GAC and WIS. Here, GAC provided a high adsorption capacity and a conductive surface for biofilm formation [11,15], while WIS contributed electron-donating properties and hydrogen generation through corrosion [20,21]. Together, this coupling of GAC and WIS reinforced refractory organic degradation, mitigated volatile fatty acids (VFAs) accumulation, and promoted direct interspecies electron transfer (DIET), ultimately driving shifts in methanogenic pathways and boosting overall process efficiency [16–18,22]."

Comments 6: For the increase in biogas would you say iron from WIS added in cell growth and enzymatic activity? while GAC increased surface area for cell attachment, resulting in high activity?

Response 6: Thank you for the valuable comments. GAC served as a microbial carrier that enriched methanogenic communities and acted as an electron conduit, thereby enhancing direct interspecies electron transfer (DIET). Concurrently, WIS regulated the pH value to alleviate the inhibition of acidified methanogenesis and supplied H₂ as an additional substrate, thus stimulating the hydrogenotrophic methanogenic pathway. Both components contributed synergistically to the enhancement of methane production.

4. Response to Comments on the Quality of English Language

Point 1: The English is fine and does not require any improvement.

Response 1: Thank you for your affirmation of me.

5. Additional clarifications

No.